Treatment of neoplasms with viruses

ABSTRACT

The subject invention relates to viruses that are able to replicate and thereby kill neoplastic cells with a deficiency in the IFN-mediated antiviral response, and their use in treating neoplastic disease including cancer and large tumors. RNA and DNA viruses are useful in this regard. The invention also relates to methods for the selection, design, purification and use of such viruses for cancer therapy.

FIELD OF THE INVENTION

[0001] The subject invention relates to viruses that are able toreplicate in and cause the death of neoplastic cells with a deficiencyin the interferon (IFN)-mediated antiviral response. RNA and DNA virusesare useful in this regard. The invention also relates to the use ofthese viruses for the treatment of neoplastic diseases including cancerand large tumors.

BACKGROUND OF THE INVENTION

[0002] Neoplastic disease which includes cancer is one of the leadingcauses of death among human beings. There are over 1.3 million new casesof cancer diagnosed in the United States each year and 550,000 deaths.Detecting cancer early, before it has spread to secondary sites in thebody, greatly increases a host's chances of survival. However, earlydetection of cancer is not always possible, and even when it is,treatments are unsatisfactory, especially in cases of highly malignantcancers. Cancer treatments, including chemotherapy and radiation, aremuch less effective in latter stages, especially when neoplastic growthsare large and/or constitute a high tumor burden. (See Hillard Stanley,Cancer Treat. Reports, Vol. 61, No. 1, Jan/Feb 1977, p.29-36, Tannock,Cancer Research, 42, 4921-4926, December 1982).

[0003] Tumor regression associated with exposure to various viruses hasbeen reported. Most of the viruses described are pathogenic in humans,and include mumps and measles. The effect of other specific viruses onparticular types of cancer cells has also been described. Smith et al,(1956) Cancer, 9, 1211 (effect of adenovirus on cervix carcinoma);Holzaepfel et al, (1957) Cancer, 10, 557 (effect of adenovirus onepithelial tumor); Taylor et al, (1970) J. Natl. Cancer Inst., 44, 515(effect of bovine enterovirus-1 on sarcoma-1); Shingu et al, (1991) J.General Virology, 72, 2031 (effect of bovine enterovirus MZ-468 onF-647a leukemia cells); Suskind et al, (1957) PSEBM, 94, 309 (effect ofcoxsackie B3 virus on HeLa tumor cells); Rukavishnikova et al, (1976)Acta Virol., 20, 387 (effect of influenza A strain on ascites tumor).

[0004] The earliest references described partial tumor regression inpatients treated with live attenuated viral vaccine with the aim tovaccinate them against smallpox or rabies. See DePace, N. G. (1912)Ginecologia, 9, 82-88; Salmon, P. & Baix (1922) Compt. Rend. Soc. Biol.,86, 819-820. Partial regression of tumors and regression of leukemiashave also been noted during naturally occurring measles infections. SeePasquinucci, G. (1971) Lancet, 1,136; Gross, S. (1971) Lancet, 1,397-398; Bluming, A. Z. and Ziegler, J. L. (1971) Lancet, 2, 105-106. Inone study of 90 cancer patients intentionally infected with live mumpsvirus, partial tumor regression was noted in 79 cases. See Asada (1994)Cancer, 34, 1907-1928. While the side effects of these viruses weretemporary, serious sequela of infection with these human pathogens is ofmajor concern.

[0005] Viruses are categorized as follows [see Murphy A and Kingsbury DW, 1990, In: Virology, 2^(nd) Edition (Ed. Fields, B. N.), Raven Press,New York, pp 9-35]: Dividing Characteristics Virus Family Names RNAviruses ss RNA, positive-sense, Picornaviridae, Caliciviridaenonsegmented, nonenveloped, ssRNA, positive-sense, Togaviridae,Flaviviridae, nonsegmented, enveloped, Coronaviridae ssRNA,negative-sense, Rhabodoviridae, Filoviridae, nonsegmented, enveloped,Paramyxoviridae ssRNA, negative-sense, Orthomyxoviridae segmented,enveloped ssRNA, ambisense, Bunyaviridae, Arenaviridae segmented,enveloped dsRNA, positive-sense Reoviridae, Birnaviridae segmented,nonenveloped ssRNA, DNA step in Retroviridae replication, positive-sense, nonsegmented, enveloped DNA viruses ss/dsDNA, nonenveloped ssDNA, nonenveloped dsDNA, nonenveloped dsDNA, enveloped HepadnaviridaeParvoviridae Papovaviridae, Adenoviridae Herpesvirdae, Poxviridae,Iridoviridae

[0006] Included among the family Herpesviridae (or Herpesviruses), arethe subfamilies Alphaherpesvirus (including Genus Varicellavirus andGenus Simpexvirus), Betaherpesvirus, and Gamimaherpesvirus.

[0007] Newcastle disease virus (“NDV”) is a member of theParamyxoviridae (or Paramyxoviruses). The natural hosts for NDV arechickens and other birds. NDV typically binds to certain molecules onthe surface of animal host cells, fuses with the cell surface, andinjects its genetic material into the host. NDV is a cytocidal virus.Once inside the cell, the viral genes direct the host cell to makecopies of the virus leading to death of the host cell, releasing thecopies of NDV which infect other cells. Unlike some viruses, NDV is notknown to cause any serious human disease. Unlike other kinds of viruses(e.g., HTLV-1, Hepatitis B), Paramyxoviruses are not known to becarcinogenic.

[0008] Temporary regression of tumors has been reported in a smallnumber of patients exposed to NDV, See Csatary, L. K. (1971) Lancet, 2,825. Csatary noted the regression of a gastrointestinal cancer in achicken farmer during an epidemic of Newcastle disease in his chickens.In a similar anecdotal report, Cassel, W. A. and Garrett, R. E. (1965)Cancer, 18, 863-868, noted regression of primary cervical cancer, whichhad spread to the lymph nodes, in a patient following injection of NDVinto the cervical tumor. Since the mechanism of tumoricidal activity wasthought to be immunologic, no work was carried out to address directtumor cytotoxicity of the virus. Instead, efforts focused upon theimmuno-modulating effects of NDV. See for example, Murray, D. R.,Cassel, W. A., Torbin, A. H., Olkowski, Z. L., & Moore, M. E. (1977)Cancer, 40, 680; Cassel, W. A., Murray, D. R., & Phillips, H. S. (1983)Cancer, 52, 856; Bohle, W., Schlag, P J., Liebrich, W., Hohenberger, P.,Manasterski, M., M ^ ller, P., and Schirrmacher, V. (1990) Cancer,66,1517-1523.

[0009] The selection of a specific virus for tumor regression was basedon serendipity or trial and error in the above citations. Only recently,have rational, mechanism-based approaches for virus use in cancertreatment been developed using DNA viruses. Examples of this type ofapproach are found in the development of recombinant adenoviral vectorsthat replicate only in tumors of specific tissue origin (Rodriguez, R.et al, 1997 Cancer Res., 57:2559-2563), or those that lack certain keyregulatory proteins (Bischoff, J R., et al, 1996 Science, 274:373-376).Another recent approach has been the use of a replication-incompetentrecombinant adenoviral vector to restore a critical protein functionlost in some tumor cells (Zhang, W W, et al, 1994 Cancer gene therapy,1:5-13). Finally, herpes simplex virus has also been engineered toreplicate preferentially in the rapidly dividing cells that characterizetumors (Mineta, T., et al, 1994 Cancer Res., 54:3963-3966).

[0010] U.S. application Ser. No. 08/260,536, hereby incorporated byreference in its entirety, discloses the use of NDV or otherParamyxovirus in the treatment of cancer.

[0011] Viral IFN Transgene Expression

[0012] One common approach to the treatment of cancer with viraltherapeutics has been the use of virus vectors for the delivery ofcertain genes to the tumor mass.

[0013] Recombinant adenovirus, adeno-associated virus, vaccinia virusand retroviruses have all been modified to express an interferon genealone or in combination with other cytokine genes.

[0014] In Zhang et al. ((1996) Proc. Natl. Acad. Sci. USA 93:4513-4518),a recombinant adenovirus expressing a human interferon consensus (i.e.,synthetic) gene was used to treat human breast cancer (and other)xenografis in nude mice. The authors concluded “. . . a combination ofviral oncolysis with a virus of low pathogenicity, itself resistant tothe effects of IFN and IFN gene therapy, might be a fruitful approach tothe treatment of a variety of different tumors, in particular breastcancer.” In contrast to subject invention which relates tointerferon-sensitive viruses, Zhang et at (1996) teach the use of aninterferon-resistant adenovirus in the treatment of tumors.

[0015] In Zhang et al. ((1996) Cancer Gene Ther., 3:31-38),adeno-associated virus (AAV) expressing consensus IFN was used totransduce human tumor cells in vitro followed by injection into nudemice. The transduced tumors either did not form tumors or grew slowerthan the non-transduced controls. Also, injection of one transducedhuman tumor cell into the tumor mass of another, non-transduced tumorresulted in a small decrease in size.

[0016] In Peplinski et al. ((1996) Ann. Surg. Oncol., 3:15-23), IFNgamma (and other cytokines, expressed either alone, or in combination)were tested in a mouse breast cancer model. Mice were immunized withtumor cells virally modified with recombinant vaccinia virus. Whenre-challenged with tumor cells, the mice immunized with virally modifiedcells had statistical improvement in the disease-free survival time.

[0017] Gastl, et al. ((1992) Cancer Res., 52:6229-6236), used IFNgamma-expressing retroviral vectors to transduce renal carcinoma cellsin vitro. These cells were shown to produce higher amounts of a numberof proteins important for the function of the immune system.

[0018] Restifo et al. ((1992) J. Exp. Med., 175:1423-1431), used IFNgamma-expressing retroviral vector to transduce a murine sarcoma cellline allowing the tumor cell line to more efficiently present viralantigens to CD8+T cells. Howard, et al. ((1994) Ann. NY Acad. Sci.,716:167-187), used IFN gamma-expressing retroviral vector to transducemurine and human melanoma tumor cells. These cells were observed toincrease the expression of proteins important to immune function. Thesecells were also less tumorigenic in mice as compared to thenon-transduced parent line, and resulted in activation of atumor-specific CTL response in vivo.

[0019] Use of Therapeutic Doses of Interferon as an Adjuvant to ViralCancer Therapy

[0020] Because of the known immune-enhancing properties of IFN, severalstudies have examined the use of IFN protein in combination with otherviral cancer vaccine therapies.

[0021] In Kirchner et al. ((1995) World J. Urol., 13:171-173), 208patients were immunized with autologous, NDV-modified, and lethallyirradiated renal-cell carcinoma tumor cells, and were co-treated withlow dose IL-2 or IFN alpha. The authors stated that this treatmentregime results in an improvement over the natural course in patientswith locally-advanced renal-cell carcinoma. The dose was approximately3.3×10³ to 2.2×10⁵ PFU/kg. This was a local therapy, as opposed to asystemic approach, with the goal of inducing an anti-tumor immuneresponse.

[0022] Tanaka et al. ((1994) J. Immunother. Emphasis Tumor Immunol.,16:283-293), co-administered IFN alpha with a recombinant vaccinia virusas a cancer vaccine therapy model in mice. This study showed astatistical improvement in survivability in mice receiving IFN ascompared to those that did not. The authors attributed efficacy of IFNto the induction of CD8-positive T cells in those animals.

[0023] Arroyo et al. ((1990) Cancer Immunol. Immunother., 31:305-311)used a mouse model of colon cancer to test the effect of IFN alphaand/or IL-2 co-therapy on the efficacy of a vaccinia virus colononcolysate (VCO) cancer treatment. They found that the triple treatmentof VCO+IL-2+IFN was most efficacious in this murine model. This approachrelies on immunization as the mechanism of anti-tumor activity

[0024] IFN was used in these studies to augment the ability of thecancer cells to be recognized by the immune system.

OBJECTS OF THE INVENTION

[0025] It is an object of the invention to provide viruses for thetreatment of diseases including cancer.

[0026] It is a further object of the invention to provide viruses forthe treatment of neoplastic diseases including cancer.

[0027] It is a further object of the invention to provide a means bywhich candidate viruses are selected and/or screened for use in thetherapy of neoplastic diseases.

[0028] It is a further object of the invention to provide guidance inthe genetic engineering of viruses in order to enhance their therapeuticutility in the treatment of neoplastic diseases.

[0029] It is a further object of this invention to provide a means withwhich to screen potential target cells for viral therapy with the goalof assessing the sensitivity of the candidate target cells to viralkilling.

[0030] It is a still further object of this invention to provideguidance in the management of viral therapy.

[0031] It is an object of the invention to provide a method for treatinglarge tumors.

[0032] It is a further object of the invention to provide purified virusand methods for obtaining same.

SUMMARY OF THE INVENTION

[0033] This invention relates to a method of infecting a neoplasm in amammal with a virus comprising administering an interferon-sensitive,replication-competent clonal virus, selected from the group consistingof RNA viruses and the DNA virus families of Adenovirus, Parvovirus,Papovavirus, Iridovirus, and Herpesvirus, to the mammal.

[0034] This invention also relates to a method of infecting a neoplasmin a mammal with a virus comprising systemically administering aninterferon-sensitive, replication-competent clonal virus to the mammal.

[0035] This invention also relates to a method of treating a neoplasmincluding cancer in a mammal comprising administering to the mammal atherapeutically effective amount of an interferon-sensitive,replication-competent, clonal virus selected from the group consistingof RNA viruses, and the DNA virus families of Adenovirus, Parvovirus,Papovavirus, Iridovirus, and Herpesvirus.

[0036] This invention also relates to a method of infecting a neoplasmin a mammal with a virus comprising administering aninterferon-sensitive, replication-competent clonal vaccinia virus,having one or more mutations in one or more viral genes involved withblocking interferon's antiviral activity selected from the group ofgenes consisting of K3L, E3L and B18R, to the mammal.

[0037] The invention also relates to a method of treating a neoplasmincluding cancer in a mammal administering to the mammal atherapeutically effective amount of an interferon-sensitive,replication-competent vaccinia virus having one or more mutations in oneor more viral genes involved with blocking interferon's antiviralactivity selected from the group of genes consisting of K3L, E3L andB18R.

[0038] The invention also relates to a method of infecting a neoplasm atleast 1 cm in size with a virus in a mammal comprising administering aclonal virus, selected from the group consisting of(1) RNA viruses; (2)Hepadenavirus; (3) Parvovirus; (4) Papovavirus; (5) Herpesvirus; (6)Poxvirus; and (7) Iridovirus, to the mammal.

[0039] The invention also relates to a method of treating a neoplasm ina mammal, comprising administering to the mammal a therapeuticallyeffective amount of a clonal virus, selected from the group consistingof(1) RNA viruses; (2) Hepadenavirus; (3) Parvovirus; (4) Papovavirus;(5) Herpesvirus; (6) Poxvirus; and (7) Iridovirus, wherein the neoplasmis at least 1 centimeter in size.

[0040] The invention also relates to a method of treating a tumor in amammal, comprising administering to the mammal a therapeuticallyeffective amount of an RNA virus cytocidal to the tumor, wherein themammal has a tumor burden comprising at least 1.5% of the total bodyweight

[0041] The invention also relates to a method of screening tumor cellsor tissue freshly removed from the patient to determine the sensitivityof the cells or tissue to killing by a virus comprising subjecting thecells or tissue to a differential cytotoxicity assay using aninterferon-sensitive virus.

[0042] The invention also relates to a method for identifying a viruswith antineoplastic activity in a mammal comprising a) using the testvirus to infect i) cells deficient in IFN-mediated antiviral activity,and ii) cells competent in IFN-mediated antiviral activity, and b)determining whether the test virus kills the cells deficient inIFN-mediated antiviral activity preferentially to the cells competent ininterferon-mediated antiviral activity.

[0043] The invention also relates to a method of making viruses for usein antineoplastic therapy comprising: a) modifying an existing virus bydiminishing or ablating a viral mechanism for the inactivation of theantiviral effects of IFN, and optionally b) creating an attenuatingmutation that results in lower virulence than said existing virus.

[0044] The invention also relates to a method of controlling viralreplication in a mammal treated with a virus selected from the groupconsisting of RNA viruses, Adenoviruses, Poxviruses, Iridoviruses,Parvoviruses, Hepadnaviruses, Varicellaviruses, Betaherpesviruses, andGammaherpesviruses comprising administering an antiviral compound.

[0045] This invention also relates to a method of treating or infectinga neoplasm in a mammal comprising subjecting a sample (e.g., serum,tumor cells, tumor tissue, tumor section) from the mammal to animmunoassay to detect the amount of virus receptor present to determineif the neoplasm will allow the virus to bind and cause cytolysis, and ifthe receptor is present, administering an interferon-sensitive,replication competent clonal virus, which binds the receptor, to themammal.

[0046] The invention also relates to a method of infecting a neoplasm ina mammal with a virus comprising systemically administering adesensitizing dose of an interferon-sensitive, replication-competentclonal virus to the mammal.

[0047] The invention also relates to a method of infecting a neoplasm ina mammal with a virus comprising administering an interferon-sensitive,replication-competent clonal virus to the mammal over a course of atleast 4 minutes.

[0048] This invention also relates to a method of infecting a neoplasmin a mammal with a virus comprising administering areplication-competent clonal virus selected from the group consisting ofthe Newcastle disease virus strain MIK107, Newcastle disease virusstrain NJ Roakin, Sindbis virus, and Vesicular stomatitis virus.

[0049] Included in the invention are:

[0050] i) a Paramyxovirus purified by ultracentrifugation withoutpelleting;

[0051] ii) a Paramyxovirus purified to a level of at least 2×10⁹ PFU permg of protein;

[0052] iii) a Paramyxovirus purified to a level of at least 1×10¹⁰ PFUper mg of protein;

[0053] iv) a Paramyxovirus purified to a level of at least 6×10¹⁰ PFUper mg of protein;

[0054] v) an RNA virus purified to a level of at least 2×10⁹ PFU per mgof protein;

[0055] vi) an RNA virus purified to a level of at least 1×10¹⁰ PFU permg of protein;

[0056] vii) an RNA virus purified to a level of at least 6×10¹⁰ PFU permg of protein;

[0057] viii) a cytocidal DNA virus which is interferon-sensitive andpurified to a level of at least 2×10⁹ PFU/mg protein;

[0058] ix) a replication-competent vaccinia virus having a) one or moremutations in one or more of the K3L, E3L and B18R genes, and b) anattenuating mutation in one or more of the genes encoding thymidinekinase, ribonucleotide reductase, vaccinia growth factor, thymidylatekinase, DNA ligase, dUTPase;

[0059] x) a replication-competent vaccinia virus having one or moremutations in two or more genes selected from the group consisting ofK3L, E3L, and B18R

[0060] xi) a Herpesvirus having a modification in the expression of the(2′-5′)A analog causing the Herpesvirus to have increased interferonsensitivity; and

[0061] xii) a Reovirus having an attenuating mutation at omega 3 causingsaid virus to become interferon-sensitive.

[0062] Also included in the invention are the following methods:

[0063] i) a method of purifying an RNA virus comprising the steps of a)generating a clonal virus; and b) purifying said clonal virus byultracentrifugation without pelleting; or c) purifying said clonal virusby tangential flow filtration with or without subsequent gel permeationchromotagraphy, and

[0064] ii) a method of purifying a Paramyxovirus comprising purifyingthe virus by ultracentrifugation without pelleting, or by tangentialflow filtration with or without subsequent gel permeationchromotagraphy.

[0065] The invention also relates to a method of treating a disease in amammal, in which the diseased cells have defects in aninterferon-mediated antiviral response, comprising administering to themammal a therapeutically effective amount of an interferon-sensitive,replication-competent, clonal virus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0066]FIG. 1 shows the effect of anti-interferon-beta antibody on viralantigen expression and infectious titer in NHEK (normal human epithelialkeratinocytes) cells.

[0067]FIG. 2 shows the effect of interferon-beta on viral antigenexpression in different cells (normal human skin fibroblasts CCD922-skand two types of head and neck carcinoma cells (KB and Hep2 cells).

[0068]FIG. 3A shows the effect of interferon on viral antigen expressionin CCD922-sk cells, and FIG. 3B shows the effect of interferon on viralantigen expression in KB cells.

[0069]FIG. 4 shows the survival curves for athymic mice bearing humanES-2 ovarian carcinoma cells and treated with either saline or NDVstrain PPMK107.

[0070]FIG. 5 shows the interferon responsiveness of a number of humantumor and normal cell lines.

DETAILED DESCRIPTION OF THE INVENTION

[0071] The present invention relates to the discovery of a novelmechanism by which viral replication selectively kills neoplastic cellsdeficient in an interferon (IFN)-mediated anti-viral response. Thisinvention also provides methods for selection, design, purification, anduse of viruses for the treatment of neoplastic diseases including cancerand large tumors. The viruses of the invention selectively replicate inand kill neoplastic cells based on the selective deficiency in thesecells of an IFN-mediated anti-viral response. Administration of theappropriate dosage of virus results in neoplastic cell death, whereasnormal cells, which possess an intact IFN-mediated anti-viral response,limit the replication of the virus and are not killed.

[0072] Included in the subject of the invention is the use ofparamyxoviruses such as NDV, and other viruses, for use in the treatmentof diseases including neoplastic disease such as cancer. The inventionalso teaches screening and engineering of other viruses suitable for useas therapeutics of neoplastic diseases. Another embodiment of theinvention involves a method of identifying tumor tissues that arecandidates for viral therapy. Finally, the invention also describes thepreparation of highly purified virus.

[0073] Rationale for the Use of Interferon-sensitive Viruses IncludingNDV to Treat Neoplastic Disease

[0074] NDV Demonstrates Selective Killing of Tumor Cells

[0075] Newcastle disease virus causes selective cytotoxic effectsagainst many human tumor cells with markedly less effects on most normalhuman cells. In a differential cytotoxicity assay, human cancer cellsderived from sarcomas, melanomas, breast carcinomas, ovarian carcinomas,bladder carcinomas, colon carcinoma, prostate carcinoma, small cell andnon-small cell lung carcinomas, and glioblastomas were discovered o beapproximately 3 to 4 orders of magnitude more sensitive to NDV than manynormal human cells [renal epithelial cells, fibroblasts, keratinocytes,melanocytes, and endothelial cells (see Example 1)]. The differentialcytotoxicity assay can also be applied to fresh isolates from thepatient's cells or tumor tissue.

[0076] An in vitro assay is used to define the tumoricidal activity ofNDV as described in Example 1. The assay measures the amount of virusrequired to kill 50% of the tested cell culture in a five day timeperiod. Examples 2 and 3 show the results of in vivo experiments inwhich virus was administered to athymic mice bearing human tumorxenografts by either the intratumoral (Example 2) or intravenous(Example 3) route. These results demonstrate that NDV can causeregression of a variety of human tumor types in a standard animal modelfor the testing of potential chemotherapeutic agents.

[0077] Evidence that NDV is specifically replicating within the tumorwas demonstrated by immunohistochemical staining for virus antigen(Example 2). Within 30 minutes of intratumoral virus injection, thetumor tissue was negative for viral antigen. However, by day 2 posttreatment, intense immunostaining for viral antigen was seen within thetumor, indicating virus replication within the tumor. Importantly, virusreplication was specific for the tumor tissue since the neighboringconnective tissue and skin was negative for viral antigen.

[0078] Importantly, efficient replication of NDV is crucial for theability of the virus to kill infected cells, as demonstrated in studiesusing UV-inactivated non-clonal virus (Lorence, R., et al, 1994 J NatlCancer Inst, 86: 1228-1233).

[0079] NDV can also cause regression of large tumors after intratumoraland intravenous administration (Examples 4 through 9). Intratumoral NDVtreatment of large intradermal A375 human melanoma xenografts (≧10 mm inmaximal dimension; tumor volume of ≧300 mm³) in athymic mice lead tohigh rates of tumor regression (Examples 4 through 8). Intravenous NDVtreatment of large subcutaneous HT1080 human fibrosarcoma xenografts(≧10 mm in maximal dimension) in athymic mice lead to complete orpartial tumor regression in five out of six mice (Example 9).

[0080] The Class I Interferon Family of Cytokines are Important NegativeModulators of Viral Infection

[0081] The class I interferons consist of the IFNα, found primarily incells of hematopoietic origin, and IFNβ found primarily in fibroblastsand epithelial cells. [Joklik, W. K. 1990. Interferons. pp. 383-410.Virology, second edition, edited by B. N. Fields, D. M. Knipe et al,Raven Press Ltd., New York; and Sreevalsan, T. 1995. Biological Therapywith Interferon-α and β: Preclinical Studies. pp. 347-364. BiologicTherapy of Cancer, second edition, edited by V. T. DeVita, Jr., S.Hellman, and S. A. Rosenberg, J. B. Lippincott Company, Philadelphia.]Both types of IFN function through an apparently common mechanism ofaction that includes the degradation of double-stranded RNAintermediates of viral replication, and the inhibition of cellulartranslation through the activity of a protein kinase activated bydouble-stranded RNA (Joklik, W. K. 1990. Interferons. pp. 383-410.Virology. Second Edition, edited by B. N. Fields, D. M. Knipe et al.,Raven Press Ltd., New York; and references therein). Several viruses(influenza, EBV, SV40, adenovirus, vaccinia) have evolved mechanisms bywhich one or more pathways of the IFN system are inactivated, thusallowing the efficient replication of the virus (Katze, M. G. 1995.Trends in Microbiol. 3:75-78).

[0082] A Wide Variety of Tumor Cells are Deficient in the Ability toLimit Viral Infection Through an IFN-dependent Mechanism

[0083] Human cervical carcinoma cells (HeLa) were overthree-hundred-fold less sensitive to the inhibition of vesicularstomatitis virus replication following pre-treatment with IFN than anon-transformed fibroblast control cell line (Maheshwari R. K., 1983.Biochem, Biophys. Res. Comm. 17:161-168). The subject inventors havediscovered that infection of a co-culture of tumorigenic human head andneck carcinoma cells (KB) and normal human skin fibroblast cells(CCD922-sk) results in viral replication initially in both cell types,followed by a limiting of the infection in the normal cells versuscontinued replication and killing of the tumor cells (Example 10).Moreover, although IFN was being secreted by the normal cells into theculture medium, the tumor cells were unable to respond to the IFN at theconcentrations being produced to establish an antiviral state. Furtherevidence for the role of IFN in the differential sensitivity of tumorcells versus normal cells to killing by NDV was obtained in two separateexperiments in which normal fibroblast cells (CCD922-sk) or normalepithelial keratinocyte cells (NHEK) were shown to become more sensitiveto infection with NDV in the presence of neutralizing antibody to IFN(Examples 11 and 12). Finally, parallel infection of normal fibroblasts(CCD922-sk) and human tumor cells (KB) in the presence of IFN revealedthat the normal cells were at least 100-fold more sensitive to theantiviral effects of added IFN than were the tumor cells (Examples 13and 14). Similar testing of variety tumor cell lines (total of 9)revealed a clear correlation in the relative sensitivity of a cell lineto killing by NDV and an inability of the cell line to manifest aninterferon-mediated antiviral response (Example 26).

[0084] Interferon and Cell Growth

[0085] There are several species of interferon (IFN) including naturaland recombinant forms of α-IFN, β-IFN, ω-IFN, and γ-IFN as well assynthetic consensus forms (e.g., as described in Zhang et al. (1996)Cancer Gene Therapy, 3:31-38). In addition to the anti-viral activitiesthat lead to its discovery, IFN is now known to play an important rolein the normal regulation of cell growth and differentiation. IFN isviewed as a negative growth regulator and several key proteins involvedin the function and regulation of IFN activity have been shown to act astumor-suppresser proteins in normal cells (Tanaka et al, 1994 Cell77:829-839). Moreover, several other proteins known to antagonize theanti-viral activity of IFN have been shown to have oncogenic potentialwhen expressed inappropriately (see below, Barber, GN, 1994, Proc. Natl.Acad. Sci. USA 91:4278-4282). Cells derived from a number of humancancers have been shown to be deleted in the genes encoding IFN (James,C D, et al, 1991, Cancer Res 51:1684-1688), and partial or complete lossof IFN function has been observed in human cervical carcinoma(Petricoin, E, et al, 1994 Mol. Cell. Bio., 14:1477-1486), chroniclymphocytic leukemia (Xu, B., et al, 1994, Blood, 84:1942-1949), andmalignant melanoma cells, (Linge, C., et al, 1995, Cancer Res.,55:4099-4104).

[0086] The IFN-inducible protein kinase (p68) has been shown to be animportant regulator of cellular and viral protein synthesis. Acorrelation has emerged that links the expression or activity of the p68kinase to the cellular state of differentiation. Thus, poorlydifferentiated cells, such as those occurring in many cancers, aredeficient in p68 function (Haines, G. K., et al, 1993 Virchows Arch BCell Pathol. 63:289-95). Cells that lack p68 activity are generallysensitive to viral mediated killing because the p68 kinase is animportant effector of the IFN-inducible antiviral state. The antiviralactivity of p68 can be antagonized through a direct interaction with acellular protein identified as p58. When cloned and overexpressed inNIH3T3 cells, p58 causes the cells to exhibit a transformed phenotypeand anchorage-independent growth (Barber G N et al., 1994 Proc Natl AcadSci USA 91:4278-4282), and a number of human leukemia cell lines havebeen shown to overexpress the p58 protein (Korth M J, et al., 1996 Gene170:181-188). Sensitivity to viral killing in undifferentiated cells canbe reversed through the induction of a more differentiated phenotype(Kalvakolanu, D V R and Sen, G. C. 1993 Proc Natl Acad Sci USA90:3167-3171).

[0087] Definitions

[0088] Cells competent in an interferon-mediated antiviral response. Asused herein, the term “cells competent in an interferon-mediatedantiviral response” are cells which respond to low levels (e.g., 10units per ml) of exogenous interferon by significantly reducing (atleast 10-fold, more advantageously at least 100-fold, moreadvantageously at least 1000-fold, and most advantageously at least10,000-fold) the replication of an interferon-sensitive virus ascompared to in the absence of interferon. The degree of virusreplication is determined by measuring the amount of virus (e.g.,infectious virus, viral antigen, viral nucleic acid). CCD922 normalfibroblasts are cells competent in an interferon-mediated antiviralresponse.

[0089] Cells deficient in an interferon-mediated antiviral response. Asused herein, the term “cells deficient in an interferon-mediatedantiviral response” are cells which fail to meet the criteria listedabove for a cell competent in an interferon-mediated antiviral response,that is, they fail to respond to low levels (e.g., 10 units per ml) ofexogenous interferon by significantly reducing the replication of aninterferon-sensitive virus as compared to in the absence of interferon.KB oral carcinoma cells are cells deficient in an interferon-mediatedantiviral response.

[0090] Clonal. Use of the term “clonal” virus is defined hereafter asvirus derived from a single infectious virus particle and for whichindividual molecular clones have significant nucleic acid sequencehomology. For example, the sequence homology is such that at least eightindividual molecular clones from the population of virions have sequencehomology greater than 95%, more advantageously greater than 97%, moreadvantageously greater than 99%, and most advantageously 100% over 300contiguous nucleotides.

[0091] Cytocidal. As used herein, the term “cytocidal” virus refers to avirus that infects cells resulting in their death.

[0092] Desensitizing Dose. As used herein, the phrase, “desensitizingdose” refers to the amount of virus required to lessen the side effectsof subsequent doses of the virus.

[0093] Differential Cytotoxicity Assay. As used herein, the phrase“differential cytotoxicity assay” for screening tumor cells or tissueusing a virus refers to the (a) virus infection of the tumor cells andone or more control cells or tissue; (b) a determination of cellsurvivability or death for each sample (for example, by the use of a dyeindicator of cell viability as in detailed in Example 1) after one ormore days of infection; and (c) based on the results, an estimation ofthe sensitivity (for example, by IC50 determination as detailed inExample 1) of the sample to the virus compared to the control(s).

[0094] Infecting a Neoplasm. As used herein, the term “infecting aneoplasm” refers to the entry of viral nucleic acid into the neoplasticcells or tissues.

[0095] Interferon-sensitive. As used herein, the phrase“interferon-sensitive” virus (e.g., NDV) means a virus that replicatessignificantly less (at least 10-fold less, advantageously at least100-fold less, more advantageously at least 1000-fold less, and mostadvantageously at least 10,000-fold less), in the presence of interferoncompared to in the absence of interferon. This is determined bymeasuring the amount of virus (e.g., infectious virus, viral antigen,viral nucleic acid) obtained from cells competent in aninterferon-mediated antiviral response in the presence or absence of lowlevels of exogenous interferon (e.g., 10 units per ml).

[0096] Neoplasm and Neoplastic Disease. As used herein, “neoplasm” meansnew growth of tissue, including tumors, benign growths (e.g.,condylomas, papillomas) and malignant growths (e.g., cancer). As usedherein, “neoplastic disease” refers to disease manifested by thepresence of a neoplasm.

[0097] Replication Competent. As used herein, the term“replication-competent” virus refers to a virus that produces infectiousprogeny in neoplastic cells.

[0098] Substantially Free of Contaminating Egg Proteins. The term“substantially free of contaminating egg proteins” refers to a level ofvirus purity in which ovalbumin is not detectable in a Western blot asperformed by one skilled in the art by (1) using 1.7×10⁹ FU of virus perwell (3.3 cm in width) run on an SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) gel (1 mm thick); (2)transferring the viral proteins from the gel to a nitrocellulosemembrane; and (3) immunostaining for ovalbumin with the use of a rabbitanti-ovalbumin [Rabbit IgG fraction at a 1:200 dilution of a 4 mg/mlantibody concentration (from Cappel, Inc.) or equivalent polyclonalantibody].

[0099] Therapeutically effective amount. As used herein, the term“therapeutically effective amount” when referring to the treatment ofneoplastic disease refers to a quantity of virus which produces thedesired effect, e.g., cessation of neoplastic growth, tumor regression,improved clinical conditions, or increased survival.

Compounds of the Invention

[0100] A diverse group of viruses are used to selectively killneoplastic cells. Natural or engineered viruses can function as anantineoplastic agent. These viruses i) infect neoplastic cells resultingin their death; ii) are replication-competent in the neoplastic cells;and iii) are limited in killing of normal cells by the antiviral effectsof interferon.

[0101] In an advantageous embodiment of the invention, the virusespossessing the above three characteristics [(i) they infect neoplasticcells resulting in their death; (ii) they are replication-competent inthe neoplastic cells; and (iii) they are limited in killing of normalcells by the antiviral effects of interferon] also induce interferon.

[0102] In another advantageous embodiment of the invention, the virusespossessing the above three characteristics also cause regression ofhuman neoplasms; and/or are not neutralized in the target humanpopulation because of the presence of pre-existing immunity.

[0103] In another advantageous embodiment, the viruses possessing theabove three characteristics are cytocidal to tumor cells.

[0104] A Paramyxovirus (as used herein “Paramyxovirus” refers to amember of the Paramyxoviridae) can be used according to the presentinvention to treat a neoplasm including a large tumor or a host having ahigh tumor burden. The Paramyxoviridae family comprises three genera:(1) paramyxoviruses; (2) measles-like viruses (morbilli viruses); and(3) respiratory syncytial viruses (pneuemoviruses). These virusescontain an RNA genome. Use of Paramyxoviridae viruses which arecytocidal, especially paramyxoviruses, e.g., Newcastle disease virus(“NDV”) and other avian paramyxoviruses such as avian paramyxovirus type2, is an advantageous method of practicing the invention. Attenuatedstrains of these viruses are especially useful for treatment ofneoplasms in accordance with the present invention.

[0105] NDV is an especially advantageous virus according to the presentinvention. NDV is categorized into three distinct classes according toits effects on chickens and chicken embryos. “Low virulence” strains arereferred to as lentogenic and take 90 to 150 hours to kill chickenembryos at the minimum lethal dose (MLD); “moderate virulence” strainsare referred to as mesogenic and take 60 to 90 hours to kill chickenembryos at the MLD; “high virulence” strains are referred to asvelogenic and take 40 to 60 hours to kill chicken embryos at the MLD.See, e.g., Hanson and Brandly, 1955 (Science, 122:156-157), and Dardiriet al., 1961 (Am. J. Vet. Res., 918-920). All three classes are useful,advantageously, mesogenic strains of NDV such as strain MK107, strain NJRoakin, and strain Connecticut-70726. (see Examples 21-23). See, e.g.,Schloer and Hanson, 1968 (J. Virol., 2:40-47) for a listing of othermesogenic strains.

[0106] For certain purposes, it is desirable to obtain a clonal virus toensure or increase the genetic homogeneity of a particular virus strainand to remove defective interfering particles. Removal of defectiveinterfering particles by cloning allows for increased purity in thefinal product as assessed by the number of total virus particles perinfectious particle (e.g., the number of particles per PFU).

[0107] Clonal virus can be produced according to any method available tothe skilled worker. For example, plaque purification is routinelyutilized to obtain clonal virus. See, e.g., Maassab et al., In: Plotkinand Mortimer, eds. Vaccines. Philadelphia: W. B. Saunders Co., 1994,pages 78-801. Triple plaque purification is especially desirable, wherea plaque is selected at each round of purification having the desiredcharacteristics, such as a preferred size, shape, appearance, orrepresentative of the parental strain. Another means of generatingclonal virus is by recombinant DNA techniques applicable by one skilledin the art. Another means of obtaining a clonal virus applies thetechnique of limiting dilution (e.g., by adding dilutions of the virussample to give an average of one or less infectious virus particles perwell containing a monolayer of a susceptible cell).

[0108] In an advantageous embodiment of the invention, purified virus isused to treat neoplastic diseases. An advantageous method forpurification of egg derived viruses are as follows (virus is notpelleted at any step in these methods):

[0109] Purification Method A

[0110] a) generating a clonal virus (e.g., plaque purification)

[0111] b) inoculating eggs with the clonal virus

[0112] c) incubating the eggs

[0113] d) chilling the eggs

[0114] e) harvesting the allantoic fluid from the eggs

[0115] f) removing cell debris from the allantoic fluid

[0116] h) ultracentrifugation of the allantoic fluid without pelleting(e.g., using a discontinuous sucrose gradient)

[0117] In another embodiment of the invention, additional steps, addedafter the removal of the cell debris (from the allantoic fluid) andbefore ultracentrifugation, consist of:

[0118] freezing then thawing the allantoic fluid

[0119] removing contaminating material from the virus suspension (e.g.,by means of centrifugation)

[0120] In another embodiment of the invention, ultracentrifugation isaccomplished by means of a continuous flow ultracentrifuge.

[0121] One embodiment of the invention relates to a method of purifyinga replication-competent RNA virus comprising the steps of

[0122] a) generating a clonal virus, and b) purifying said clonal virusby ultracentrifugation without pelleting.

[0123] Another embodiment of the invention involves a method ofpurifying a paramyxovirus (e.g., NDV) comprising purifying the virus byultracentrifugation without pelleting. Optionally, the purifying stepadditionally comprises prior to the ultracentrifugation:

[0124] a)plaque purifying to generate a clonal virus,

[0125] b)inoculating eggs with the clonal virus,

[0126] c)incubating the eggs,

[0127] d)chilling the eggs,

[0128] e)harvesting allantoic fluid from the eggs and,

[0129] f)removing cell debris from the allantoic fluid.

[0130] Another, embodiment of the invention involves a method ofpurifying a replication-competent clonal virus from eggs or cell culturecomprising the step of ultracentrifugation without a step in which thevirus is pelleted.

[0131] Another embodiment of the invention involves a method of thepurifying a paramyxovirus (e.g., NDV) comprising purifying the virus bysequential tangential flow filtration (TFF). Optionally, the virus canbe additionally purified by gel permeation chromatography, where each ofthese steps occurs in the presence of a stabilizing buffer (Example 15):

[0132] a) plaque purifying to generate a clonal virus,

[0133] b)inoculating eggs with the clonal virus,

[0134] c)incubating the eggs,

[0135] d)chilling the eggs,

[0136] e)harvesting allantoic fluid from the eggs and dilution ofallantoic fluid with buffer,

[0137] f)removing cell debris from the allantoic fluid by TFF,

[0138] g)purification of the virus by TFF, and

[0139] h)purification of the virus by gel permeation chromatography.

[0140] Optionally, the virus obtained from the gel permeation step canbe concentrated using TFF.

[0141] Another embodiment of the invention involves a method ofpurifying a replication-competent clonal virus from eggs or cell culturecomprising the step purifying the virus by sequential tangential flowfiltration (TFF), optionally followed by gel permeation chromatography,optionally followed by TFF to concentrate the virus.

[0142] Clonal virus

[0143] Use of these methods permits purification of a clonal virus[including Paramyxovirus (e.g., NDV)] to at least 2×10⁹ PFU/mg protein,advantageously to at least 3×10⁹ PFU/mg protein, more advantageously toat least 5×10⁹ PFU/mg protein, more advantageously to at least 1.0×10¹⁰PFU/mg protein, more advantageously to at least 2.0×10¹⁰ PFU/mg protein,more advantageously to at least 3×10¹⁰ PFU/mg protein, moreadvantageously to at least 4×10¹⁰ PFU/mg protein, more advantageously toat least 5×10¹⁰ PFU/mg protein, and most advantageously at least 6×10¹⁰PFU/mg.

[0144] Use of these methods permits purification of a clonal virus[including Paramyxovirus (e.g., NDV)] to level in which the number ofvirus particles per PFU is less than 10, more advantageously less than5, more advantageously less than 3, more advantageously less than 2, andmost advantageously less than 1.2. (Lower numbers of virus particles perPFU indicate a higher degree of purity.)

[0145] RNA Viruses

[0146] In another embodiment, these methods permit purification (to thelevels cited above for clonal viruses) of an RNA virus [including (a) acytocidal RNA virus; (b) a single-stranded RNA non-segmented,nonenveloped virus; (c) a single-stranded RNA segmented, envelopedvirus; (d) a double-stranded RNA segmented, nonenveloped virus; (e) anda single-stranded RNA non-segmented, enveloped virus (e.g.,Paramyxovirus (e.g., NDV) and e.g., Retroviruses].

[0147] DNA Viruses

[0148] In another embodiment, these methods permit purification (to thelevels cited above for clonal viruses) of an interferon-sensitivecytocidal virus selected from the group consisting of (a) enveloped,double-stranded DNA viruses (including poxviruses); (b) nonenveloped,single-stranded DNA viruses; and (c) nonenveloped, double-stranded DNAviruses.

[0149] Egg Derived Viruses

[0150] In another embodiment, these methods permit purification of eggderived viruses to a level substantially free of contaminating eggproteins. It is preferred to limit the amount of egg proteins in viruspreparations for human therapeutic use since major egg proteins likeovalbumin are allergens.

[0151] Viruses useful in the treatment of neoplastic diseases includingcancer are shown in Table 1. These viruses are optionally screened fornaturally occurring variations (certain strains or isolates) that resultin altered IFN production relative to the parental strain.

[0152] In another embodiment of this invention, candidate viruses,whether naturally occurring or engineered, are tested for the ability toprovide therapeutic utility in the treatment of neoplasms. In oneembodiment, the amount of candidate virus required to kill 50% of cellsdeficient in an interferon-mediated antiviral response, e.g., KB headand neck carcinoma cells, is compared to the amount of virus required tokill 50% of a similar number of cells competent in aninterferon-mediated antiviral response, for example normal skinfibroblasts. The amount of killing is quantified by any number of meansincluding trypan blue exclusion or MTT assay (see Example 1). Asignificant reduction (e.g., at least 5-fold) in the amount of virusrequired to kill cells deficient in an interferon-mediated antiviralresponse relative to the amount needed to kill cells competent in aninterferon-mediated antiviral response indicates that the virus beingtested exhibits activity required for therapeutic utility in thetreatment of neoplasms. Other NDV viruses and Sindbis virus are suchnatural occurring viruses that display tumor-selective killing (seeExamples 21-23, and 25). TABLE 1 Naturally Occurring Viruses for Use inCancer Therapy Virus Class Virus Family Virus Example RNA, negativeParamyxoviridae Newcastle Disease Virus stranded Avian ParamyxovirusType 2 Mumps Human Parainfluenza Rhabdoviridae Vesicular StomatitusVirus RNA, positive Togaviridae Sindbis Virus stranded FlaviviridaeYellow Fever Virus (attenuated) Picomaviridae Rhinovirus BovineEnterovirus Echovirus Coronaviridae Avian Infectious Bronchitis VirusHuman Coronaviruses

[0153] An understanding of the factors involved in the establishment ofan antiviral state allows for the creation of a screening assay fortumors that are likely to respond to viral therapy. In principle,patient derived tumor tissue obtained from biopsy is screened for theexpression of p68 kinase, p58, or other factors involved in theregulation of an antiviral state or cellular differentiation. Otherfactors include, but are not limited to, interferon response factor-I(IRF-1), interferon stimulatory gene factor-3 (ISGF-3), c-Myc, c-Myb,and IFN receptors. In the case of c-Myc, c-Myb or p58, high levelexpression indicates that the tumor tissue or cells are treatmentcandidates for virus therapy. In the case of p68, IRF-I, ISGF-3, and IFNreceptors, low level expression indicates that the tumor tissue or cellsare treatment candidates for virus therapy.

[0154] In another embodiment of this invention, primary tumor tissue orcells obtained from patient biopsies are expanded in culture and testedfor sensitivity to killing by a suitable viral therapy. In oneembodiment, the amount of virus required to kill 50% of the tumor tissueculture is compared to the amount required to kill 50% of a culture ofnormal cells as described above for the screening of candidate viruses.An increase of ten-fold or greater in the sensitivity of the tumor cellsrelative to normal cells to killing by the viral agent indicates thatthe tumor cells are specifically sensitive to the cytocidal effects ofthe viral treatment. In a further embodiment of the invention, theability of the targeted tumor cells to respond to endogenously orexogenously supplied IFN is determined by conducting the above screen inthe presence of IFN (alpha or beta form, using e.g., 10 units per ml,see Example 27).

[0155] An understanding of the cellular receptors required for virusattachment or entry allows additional screening for tumors that havehigh receptor expression and hence enhanced sensitivity to theinterferon-sensitive virus. This is an additional level screening forpatients that are likely to respond to virus therapy. Advantageously fortherapy with an interferon-sensitive virus, the patient's tumor is bothresistant to interferon and has high expression of the cellular receptorfor the virus. In principle, patient derived serum, tumor cells,tissues, or tissue sections are screened by immunoassay or immunostainfor the amount of virus receptor present in the serum or on the tumorcells or tumor tissue. For example, Sindbis virus utilizes the highaffinity laminin receptor to infect mammalian cells (Wang et al., 1992,J Virol., 66, 4992-5001). This same receptor is known to be expressed inhigher amounts in many diverse types of metastatic cancer. The PANC-1renal cancer cell line, and the colon adenocarcinoma cell line SW620 areknown to express a high level of high affinity laminin receptor mRNA(Campo et al, 1992, Am J Pathol 141:107301983; Yow et al., (1988) Proc.Natl Acad Sci, 85, 6394-6398) and are highly sensitive to Sindbis virus(Example 25). In contrast, the rectum adenocarcinoma cell line SW1423 isknown to express very low levels of high affinity lamin receptor mRNA(Yow et al., (1988) Proc. Natl Acad Sci, 85, 6394-6398), and is morethan 4 orders of magnitude more resistant to killing by PPSINDBIS-Ar339than SW620 cells.

[0156] Existing strains of NDV, or other viruses including RNA and DNAviruses, are screened or engineered for altered IFN responses (e.g.,advantageously increased IFN responses) in normal cells. In addition tothe ability to elicit a strong IFN response, other viral characteristicsare screened for or engineered into the virus. Viruses with alteredreceptor specificity (e.g., Sindbis virus PPSINDBIS-Ar339, see Example25), or low neurovirulence are included in the subject invention (e.g.,NDV virus PPNJROAKIN, see Example 24). Advantageously, viruses of theinvention have the capacity to spread through direct cell to cellcontact.

[0157] The invention described herein includes a broad group of viruses(see Table 1) that are useful for treatment of neoplasms in a manneranalogous to the indication for NDV. In addition, viruses that naturallywould not be candidates for use, due to the presence of a mechanism(s)to inactivate the IFN response in normal cells, are optionallyengineered to circumvent the above restrictions. If left unmodified,viruses with mechanisms to inactivate the interferon response would bemore toxic to normal cells than viruses with such mechanism removed. Thesubject invention provides (1) the development of a vector that can beeasily manipulated; and (2) the creation of a set of therapeuticviruses. Manipulations include the addition of an IFN gene to permit theviral expression of a transgene expressing IFN, or other activators ofthe IFN response pathway. Additional permutations include the engineeredexpression of pro-drug activating enzymes such as the Herpesvirusthymidine kinase or cytosine deaminase (Blaese R M et al., 1994. Eur. J.Cancer 30A: 1190-1193) and the expression of suitable marker antigen toallow targeting of tumor cells by the immune system. An additionalpermutation include the engineered expression of receptor ligands totarget cells with those receptors [e.g., expression of receptors toother viruses to target cells infected with those viruses (seeMebastsion et al., 1997, Cell 90:841-847; and Schnell M J et al., 1997,Cell 90:849-857].

[0158] Several Newcastle Disease virus strains demonstrate selectivekilling of tumor cells. In a differential cytotoxicity assay using asecond strain of mesogenic Newcastle Disease virus, tumor cells werefound to be 3 orders of magnitude more sensitive than normal cells tokilling by the virus (Example 21). Additionally, when a third mesogenicNewcastle Disease virus strain was used in a differential cytotoxicityassay, tumor cells were found to be 80 to 5000-fold more sensitive thannormal cells to killing by the virus (Example 22). Both of thesemesogenic Newcastle Disease virus strains also caused tumor growthregression following intratumoral administration to athymic mice bearinghuman tumor xenografts (Example 23).

[0159] In separate experiments, the safety of three distinct NewcastleDisease virus strains were studied following intracerebral inoculationin athymic and immune-competent mice. The results of this study showedthat all three virus strains were well tolerated in mice with an intactimmune system. Intracerebral inoculation into the brains of athymic micerevealed that one of the viruses was tolerated significantly better thanthe other two (Example 24). These results demonstrate that within asingle virus family important differences in viral properties can occurand be can be exploited therapeutically for greater efficacy orincreased safety.

[0160] Another means by which increased efficacy and lower toxicityfollowing treatment with oncolytic viruses can be achieved is throughthe use of interferon-sensitive viruses that require specific cellsurface receptors that are preferentially expressed on tumor cells.Sindbis virus provides an example of this type of restriction. Sindbisvirus infects mammalian cells using the high affinity laminin receptor(Wang et al, (1992) J. Virol. 66, 4992-5001). When normal and tumorcells were infected with Sindbis virus in a differential cytotoxicityassay, cells which both were tumorigenic and expressed the high affinitylaminin receptor were found to be more sensitive to killing by thisvirus than other cells (Example 25). Normal keratinocytes express thehigh affinity laminin receptor (Hand et al., (1985) Cancer Res., 45,2713-2719), but were resistant to killing by Sindbis in this assay.

[0161] Vesicular Stomatitis Virus (VSV) provides evidence oftumor-selective killing of by enclitic viruses, i.e., an inherentdeficiency in interferon responsiveness in tumor cells renders thesecells sensitive to killing by interferon-sensitive replication-competentviruses. When VSV was used to infect non-tumorigenic human WISH cellsand tumorigenic HT1080 or KB cells in the presence of exogenousinterferon.

[0162] Below is a list of viruses that when modified to removenaturally-occurring anti-interferon activities, are useful for viralcancer therapy (see Table 2). Modified viruses (advantageously, but notnecessarily, attenuated in addition to the anti-interferon modification,see Table 3) that have had endogenous anti-interferon activitiesdestroyed or reduced, are useful for cancer therapy. This list includes,but is not be limited to, the viruses described below. Because of thesimilarity between viruses of a common class, the identified mechanismsfor each of the specific viruses listed below, are also present in othermembers of that class of virus as identical or functionally analogousmechanisms. The broader group of viruses is added in parenthesis.Viruses, such as those below, that have a functional loss ofanti-interferon activity, through any means, including natural occurringmutations, as well as engineered deletions or point mutations, areuseful in the methods of the subject invention.

[0163] Viruses that exercise more than one mechanism are optionallymodified to contain mutations in one, some, or all of the activities.Mutations for some of the described activities are available in thegeneral scientific community.

[0164] Isolates of naturally occurring or engineered virus that areslower growing, compared to the growth rate of wild-type virus, areparticularly advantageous because a slower virus growth rate will allowa cell or population of cells competent in an interferon response toestablish an efficient antiviral state before viral replication can killthe cell or cell population.

[0165] The disabling of viral anti-interferon activities as a specificalteration of viral character that results in the augmentation of theinterferon response in an infected cell, but still allows viralreplication in neoplastic cells is included in the subject invention.

[0166] Table 2 shows existing viruses engineered to removeanti-interferon activity. Table 3 lists viruses engineered to beattenuated in virulence. TABLE 2 Extant Viruses Engineered to RemoveAnti-IFN Activity Virus Class Virus Family Virus Anti-IFN ActivityReference RNA Reoviridae reovirus σ3 Imani F and Jacobs B (1988) ProcNatl Acad Sci USA 85:7887-7891 DNA Poxviridae Vaccinia K3L Beattie E etal. (1991) Virology 183:419 E3L Beattie E et al. (1996) Virus Genes12:89-94. B18R Symons JA et al (1995) Cell 81:551-560 Adenoviridaevarious VA₁ transcripts Mathews MB and Shenk T (1991) J Virol64:5657-5662. subtypes Alphaherpesvirinae HSV-1 gamma 34.5 Chou J et al(1996) Proc Natl Acad Sci USA 92:10516-10520 gene product

[0167] TABLE 3 Known Attenuating Mutations in Selected Viruses VirusClass Virus Family Virus Attenuation Reference RNA Reoviridae reovirusσ1 Spriggs DR and Fields BN (1982) Nature 297:68-70. rotavirus bovinestrains (WC3) Clark HF (1988) J Infect Dis 158:570-587. DNA PoxviridaeVaccinia vaccinia growth factor Buller RML et al (1988) Virology164:182. thymidine kinase Buller RML et al (1985) Nature 317:813-815.thymidylate kinase Hughes SJ et al (1991) J Biol Chem 266:20103-20109DNA ligase Kerr SM et al (1991) EMBO J 10:4343-4350. ribonucleotidereductase Child SJ et al (1990) Virology 174:625-629. dUTPase Perkus MEet al (1991) Virology 180:406-410 Adenoviridae various Ad-4, Ad-7, Ad-21Takafugi ET et al (1979) J Infect Dis 140:48-53. subtypesAlphaherpesvirinae HSV-1 thymidine kinase Field HJ and Wildy P (1978) JHyg 81:267-277. ribonucleotide reductase Goldstein DJ and Weller sk(1988) Virology 166:41-51. gamma 34.5 gene product Chou J et al (1995)Proc Natl Acad Sci USA 92:10416-10520 b′a′c′ inverted repeats Meignier Bet al (1988) J Infect Dis 162:313-322

[0168] Treatment of Neoplasms

[0169] The present invention relates to viral therapy of neoplasms,especially in animals having cancer. In an advantageous embodiment, theinvention relates to the treatment of tumors which are 1 centimeter (cm)or more in size as measured in the greatest dimension. As used herein,“a 1 cm tumor” indicates that at least one dimension of the tumor is 1cm in length. Such tumors are more sensitive than expected to viraltherapy, often at least as sensitive to virus, if not more sensitive,than tumors which are smaller in size. In a more advantageous aspect ofthe invention, tumors greater than 1 cm. are treated, e.g., tumors whichare 2 cm or greater, from about 2 cm to about 5 cm, and greater than 5cm.

[0170] The present invention can also be employed to treat hosts havinga high tumor burden. As used herein, the phrase “tumor burden” refers tothe total amount of tumor within the body expressed as a percentage asbody weight. Viral therapy of hosts having a tumor burden, e.g., fromabout 1% to about 2% of total body weight is surprisingly effective,e.g., producing tumor regression and a reduction in the overall tumorload. This is especially unexpected since a tumor burden ofapproximately 2% of the total body weight (e.g., a 1 kg tumor in a 60 kghuman) is approximately the maximum cancer mass compatible with life.See, e.g., Cotran et al, In Robbins Pathological Basis of Diseases, 4thEdition, W B Saunders, 1989, page 252. In the Examples, volumes up to397 mm³ for a melanoma cancer (e.g., A375) in a mouse host showedcomplete regression in response to treatment with a Newcastle diseasevirus (e.g., a triple-plaque purified virus). Assuming that for tissue1000 mm³ equals 1 gram, a tumor having a volume of 397 mm³ comprisesapproximately 2% of the total body weight for a 20 gram mouse.

[0171] As shown in Examples 4 to 9 below, tumor regression was achievedwith tumors at least 1 cm in size, while untreated, control animalsbegan dying from tumor burden within several weeks. Thus, such diseasedanimals were successfully treated despite being within two weeks ofdeath. Thus, in accordance with the present invention, an animal whichis near terminal from its tumor burden can be treated effectively withviral therapy. Consequently, the present invention can be used to treatpatients who have not responded to conventional therapy, e.g.,chemotherapy such as methotrexate, 5-fluorouracil, and radiationtherapy.

[0172] The efficacy of NDV for the treatment of cancer followingadministration through the intraperitoneal route has also been examined.Using an ascites prevention model of ovarian cancer, intraperitonealinjection of NDV in mice harboring ES-2 human ovarian tumors resulted inincreased survival compared to mice treated with saline (Example 16).When ES-2 cells were used in an ovarian cancer tumor model withtreatment initiated once ascites formed, ascites fluid production wasmarkedly decreased in virus-treated animals compared to saline controls(Example 17).

[0173] In another embodiment of the invention, the administration ofvirus results in 1) the relief of tumor related symptoms, such as butnot limited to deceased rate of ascites fluid production, relief ofpain, and relief of obstructive disease, and 2) the prolongation oflife.

[0174] Twenty-three patients have received the plaque purified NDVisolate by the intravenous route (Example 20). Treatment responsesinclude the regression of a palpable tumor, the stabilization of diseasein 47% of patients and a reduction in pain medication.

[0175] Administration and Formulation

[0176] In one embodiment of the invention, tumor cells or tissue arescreened in vitro to determine those patients with tumors sensitive tothe virus. Tumor cells removed from the patient (by methods such as fineneedle aspiration for solid tumors or by paracentesis for ovarianascites tumors) are grown in vitro and incubated with virus. In thisembodiment of the invention, patients are selected for therapy if thevirus has a high activity against their tumor cells.

[0177] In an advantageous embodiment of the invention, the amount ofvirus administered results in regression of the tumor or tumors. As usedherein, the term “regression” means that the tumor shrinks, e.g., insize, mass, or volume. Shrinkage in tumor size is demonstrated byvarious methods, including physical examination, chest film or otherx-ray, sonography, CT scan, MRI, or a radionucleotide scanningprocedure.

[0178] Various types of neoplasms including cancers are treatable inaccordance with the invention. The viruses of the present invention areuseful to treat a variety of cancers, including but not limited to lungcarcinoma, breast carcinoma, prostate carcinoma, colon adenocarcinoma,cervical carcinoma, endometrial carcinoma, ovarian carcinoma, bladdercarcinoma, Wilm's tumor, fibrosarcoma, osteosarcoma, melanoma, synovialsarcoma, neuroblastoma, lymphoma, leukemia, brain cancer includingglioblastoma, neuroendocrine carcinoma, renal carcinoma, head and neckcarcinoma, stomach carcinoma, esophageal carcinoma, vulvular carcinoma,sarcoma, skin cancer, thyroid pancreatic cancer, and mesothelioma. Theviruses of the present invention are also useful to treat a variety ofbenign tumors, including but not limited to condylomas, papillomas,meningiomas, and adenomas.

[0179] A therapeutically effective amount of virus is administered to ahost having a neoplasm. It is understood by those skilled in the artthat the dose of virus administered will vary depending on the virusselected, type of neoplasm, the extent of neoplastic cell growth ormetastasis, the biological site or body compartment of the neoplasm(s),the strain of virus, the route of administration, the schedule ofadministration, the mode of administration, and the identity of anyother drugs or treatment being administered to the mammal, such asradiation, chemotherapy, or surgical treatment. These parameters aredefined through maximum tolerated dose determination in animal modelsand scaling to human dosage as a function of relative body surface areaor body mass. It is also understood that under certain circumstances,more than one dose of the virus is given. The optimal interval betweensuch multiple doses of the virus can be determined empirically and iswithin the skill of the art. NDV is generally administered from about3×10⁶ to about 5×10¹² PFU of virus. For local administration (e.g.,directly into a tumor), total amounts of from about 3×10⁶ to about5×10¹⁰ PFU of virus are typically used. For systemic administration,amounts of from about 1×10⁸ to about 4×10¹¹ PFU of virus per squaremeter of body surface area are used. For intravenous administration,dosing schedules of once per week, two times per week and three timesper week are used. A virus in accordance with the present invention,optionally with a chemotherapeutic agent, can be administered by variousroutes, e.g., enteral, parenteral, oral, nasal, rectal, intrathecal,intervenous (e.g., using a catheter), subcutaneous, intratumor (e.g.,directly into its tissue or into vessels which perfuse it), peritumoral,local, sublingual, buccal, topical, intramuscular, by inhalation,percutaneous, vaginal, intra-arterial, intra-cranial, intradermal,epidural, systemically, topical, intraperitoneal, intrapleural, etc. Forlung tumors, a bronchial route (e.g., bronchial administration) can beused. Endoscopic injections of gastrointestinal tumors, as well assuppository treatments of rectal tumors are also used where appropriate.

[0180] Murine toxicity studies with NDV have indicated that the acutetoxicity following intravenous virus administration is likely to becaused by cytokine mediated reactions. Cytokine responses to repeatedstimuli are known to be desensitized, or down-regulated, following theinitial induction event (Takahashi et al., (1991) Cancer Res. 51,2366-2372). Mice intravenously injected with a desensitizing dose ofvirus were able to tolerate approximately 10-fold more virus on a seconddose than mice receiving vehicle alone for the first injection (Example18).

[0181] The rate of virus administration by the intravenous route cansignificantly affect toxicity.

[0182] Two groups of athymic mice were intravenously treated withidentical doses of NDV which was administered either slowly (0.2 ml over4 minutes) or rapidly (0.2 ml over 30 seconds). Comparison of themaximal weight lose in each group revealed 50% less weight loss in thegroup receiving slow injection versus a rapid injection (Example 19).

[0183] In one cohort of a clinical trial, patients received threeinjections of the plaque purified NDV isolate over the course of oneweek (Example 20). Under these conditions, a desensitizing effect of theinitial dose lessened the toxicity associated with the second and thirddoses. These data parallel those obtained with the animal studies shownin Example 18. One concern related to the use of oncolytic viruses inthe treatment of cancer is the potential inhibitory effect the humoralimmune response can exert on the therapy. In the clinical study,patients displaying stable disease after 1 month are eligible for asecond course of treatment which then is administered in the presence ofneutralizing antibodies to NDV. Nevertheless, infectious virus could befound in patient urine seven days after dosing for the second course,providing evidence that administration of high doses of virus canovercome the effect of neutralizing antibodies and establish aninfection within the patient.

[0184] In an advantageous embodiment of the invention, a desensitizingdose is given before higher subsequent doses. For desensitization, virusdoses of 1×10⁸ to 2.4×10¹⁰ PFU/m² are used. After desensitization,additional virus doses of 3×10⁸ to 4×10¹² PFU/m² are used. The timeframe between doses, including the time frame between desensitizing doseand the next dose, is 1 to 14 days, advantageously 1 to 7 days. Thedesensitizing dose can be administered by various routes, e.g.,intravenous, enteral, parenteral, oral, nasal, rectal, intrathecal,intervenous, subcutaneous, intratumor, peritumoral, local, sublingual,buccal, topical, intramuscular, by inhalation, percutaenous, vaginal,intra-arterial, intracranial, intradermal, epidural, sytemically,topical, intraperitoneal, intrapleural, endoscopic, intrabronchial, etc.The subsequent doses can be administered by the same route as thedesensitizing dose or by another route, e.g., intravenous, enteral,parenteral, oral, nasal, rectal, intrathecal, intervenous, subcutaneous,intratumor, peritumoral, local, sublingual, buccal, topical,intramuscular, by inhalation, percutaenous, vaginal, intra-arterial,intracranial, intradermal, epidural, sytemically, topical,intraperitoneal, intrapleural, endoscopic, intrabronchial, etc.

[0185] Optionally, more than one route of administration can be used ineither a sequential or concurrent mode. Routes for either concurrent orsequential administration include but are not limited to intravenous,enteral, parenteral, oral, nasal, rectal, intrathecal, intervenous,subcutaneous, intratumor, peritumoral, local, sublingual, buccal,topical, intramuscular, by inhalation, percutaenous, vaginal,intra-arterial, intracranial, intradermal, epidural, sytemically,topical, intraperitoneal, intrapleural, endoscopic, intrabronchial, etc.An example would be the administration of a intravenous desensitizingdose followed by an intraperitoneal dose.

[0186] In another advantageous embodiment of the invention, the virus isadministered by slow infusion including using an intravenous pump orslow injection over the course of 4 minutes to 24 hours.

[0187] A virus, and optionally one or more chemotherapeutic agents, isadministered by a single injection, by multiple injections, orcontinuously. The virus is administered before, at the same time, orafter the administration of chemotherapeutic agents (such as but notlimited to: busulfan, cyclophosphamide, methotrexate, cytarabine,bleomycin, cisplatin, doxorubicin, melphalan, mercaptopurine,vinblastine, 5-fluorouracil, taxol, and retinoic acid). Viral therapy inaccordance with the present invention is optionally combined with othertreatments, including, surgery, radiation, chemotherapy (see, e.g.,Current Medical Diagnosis and Treatment, Ed. Tierney et al, Appleton &Lange, 1997, especially pages 78-94), and biological therapy. The virusis administered before, at the same time, or after the administration ofbiological agents such as (1) other oncolytic agents [such as but notlimited to: adenoviruses with one of its genes under transcriptionalcontrol of a prostate cell specific response element (see Rodrigues, R.et al, 1997, Cancer Res, 57:2559-2563; adenoviruses which do not encodea Elb polypeptide capable of binding p53 (see Bischoff, J. R., et al,1996, Science 274:373-376); a herpes simplex virus that is incapable ofexpressing 99 functional gamma 34.5 gene product (see Mineta, T. et al,1995, Nature Medicine, 1:938-943)]; (2) cytokines (such as but notlimited to: colony stimulating factors such as GM-CSF; tumor necrosisfactor, and interleukins such as IL-I, IL-2, IL-6 and IL-10); (3) viralvectors [such as but not limited to adenovirus encoding p53 (see Zhang,W W et al, 1994, Cancer Gene Therapy. 1:5-13)]; and (4) cancer vaccines.

[0188] In one embodiment of the invention, therapy consists of theserial treatment with antigenically distinct viruses which are cytotoxicand tumor selective via the IFN mechanism. This embodiment allows viraltherapy over an extended period without immunological interference.

[0189] Another embodiment involves the treatment of patients with IFN(e.g. αIFN, βIFN or γIFN) prior to, concurrent with, or followingadministration of NDV (or other virus). The IFN is selected from thegroup class I (alpha, beta and omega) and class II (gamma), andrecombinant version and analogs thereof as discussed in, for example,Sreevalsoun, T., 1995 (In: Biologic Therapy of Cancer, second edition,edited by V. T. DeVita, Jr., S. Hellman, and S. A. Rosenberg, J. B.Lippincott Company, Philadelphia, pp347-364). Normal cells respond tothe IFN pre-treatment with an augmented IFN response to viral infectionaffording even greater safety to these cells. Tumor cells deficient inthe IFN signaling pathway remain sensitive to killing by the virus. Thisallows even higher doses of viral therapy to be used. The IFN isadministered in accordance with standard clinical guidelines for dosesand regimens known to be effective for treating viral infections. Inanother embodiment of the invention, other drugs, known to affect theIFN response pathway are also optionally used to increase thesensitivity of tumor cells, or increase the resistance of normal cellsto the cytocidal effects of viral infection. This class of drugsincludes, but is not limited to tyrosine kinase inhibitors, cimetidine,and mitochondrial inhibitors. Hypoxia and hyperthermia are also known tomodulate interferon responsiveness.

[0190] In another embodiment of the invention, immunosuppressants suchas cyclosporin A, azathiaprime, and leflunomide, various corticosteroidpreparations, and anti-CD-40 ligand antibodies (Foy, T. M., et al.,1993, J. Exp. Med. 178:1567-1575) are administered with the virus.Alternatively, an immunostimulatory compound, e.g., lipopeptides, can beadministered with the virus.

[0191] An independent mechanism by which the amount of interferonproduced in response to viral infection is increased through the use ofnucleosides (Machida, H., 1979. Microbiol. Immunol. 23:643-650),nucleoside precursors, or drugs that increase the cellular concentrationof one or more nucleosides, are optionally used as an adjunct to viraltherapy.

[0192] Certain purine nucleoside analogs, e.g., 2-chlorodeoxyadenosineand 2′-deoxycoformycin, reduce interferon production in vivo. Suchcompounds are used to further effect differences in interferonsensitivities of tumor cells versus normal cells and are optionally usedas an adjunct to viral therapy.

[0193] In one aspect, an effective amount of virus can be subdividedinto smaller dose units and injected at the same time into differentlocations of the same tumor. For continuous administration, the desiredagent(s) is administered via an implanted minipump or it is impregnatedinto a desired polymer and then transplanted into a desired location(e.g., directly into the tumor) for slow or delayed release.

[0194] A virus of the present invention is formulated as apharmaceutical preparation by bringing it into a suitable dose form,together with at least one excipient or auxiliary, and, if desired, withone or more further active compounds. The preparations are utilized inboth human and veterinary medicine. Suitable excipients include, e.g.,organic and inorganic substances which are appropriate for enteral orparenteral administration, e.g., water, saline, tissue culture media,buffers, lysine, citrate, glycerol triacetate and other fatty acidglycerides, gelatin, soya lecithin, carbohydrates such as, mannitol,sucrose, lactose or starch, magnesium stearate, talc, cellulose orprotein carriers, or a combination of the preceding compounds, such asmannitol/lysine, or mannitol/lysine/sucrose. The preparations aresterilized and/or contain additives, such as preservatives orstabilizers. For parenteral administration, e.g., systemic or localinjection, a virus preparation is formulated, e.g., as an aqueoussuspension or emulsion.

[0195] The invention also relates to a method of treating a disease in amammal, in which the diseased cells have defects in aninterferon-mediated antiviral response, comprising administering to themammal a therapeutically effective amount of an interferon-sensitive,replication-competent, clonal virus. For example, cells infected withmany viruses like hepatitis B that disable the interferon response aresusceptible to the viruses of this invention. There is evidence thathuman immunodeficiency virus (HIV) disables the interferon response. Theinterferon-sensitive viruses of this invention are useful in treatingsuch chronic virus infections such as those due to hepatitis B,hepatitis C, HIV, Epstein-Barr virus, human papilloma virus, and herpesvirus.

[0196] Unless indicated otherwise herein, details and conditions ofviral therapy of this invention are in accordance with U.S. applicationSer. No. 08/260,536 whose disclosure is incorporated herein by referencein its entirety. The entire disclosure of all applications, patents andpublications, cited above and in the figures are hereby incorporated byreference.

[0197] The following examples are illustrative, but not limiting of themethods and compositions of the present invention. Other suitablemodifications and adaptations of a variety of conditions and parametersnormally encountered in clinical therapy which are obvious to thoseskilled in the art are within the spirit and scope of this invention.

EXAMPLE 1

[0198] PPMK107, (a Triple Plaque Purified Isolate of the NDV StrainMK107) Demonstrates a Selective Cytotoxic Activity toward many HumanCancer Cells Compared to Normal Human Cells

[0199] Human tumor cells and normal cells were grown to approximately80% confluence in 24 well tissue culture dishes. Growth medium wasremoved and PPMK107 was added in 10 fold dilutions ranging from 10⁶plaque forming units (PFU)/well to 10⁻¹ PFU/well. Controls wells with novirus added were included on each plate. Virus was adsorbed for 90minutes on a rocking platform at 37° C. At the end of the incubationperiod, the viral dilutions were removed and replaced by 1 ml of growthmedium. Plates were then incubated for 5 days at 37° C. in 5% C02, thenassessed qualitatively for the amount of cytopathic effect (CPE).Cytotoxicity was quantified by using a colorimetric MTT(2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay(Cell Titer 96, catalog #G4000, Promega Corporation, Madison Wis. 53711)monitored at 570 nm, that detects mitochondrial enzyme activity (Mosman,T., 1983, J. Immunol. Methods 65:55). The viability in the virus treatedwells was expressed as a percent of the activity in untreated controlwells. The data was plotted graphically as PFU/well vs. viability as apercent of control. The IC50 was calculated as the amount of virus inPFU/well causing a 50% reduction in the amount of viable cells.

[0200] The results are given in Tables 4, 5 and 6. PPMMK107 demonstrateda high degree of cytotoxic activity against a diverse set of humancancer cells with 30 out of 39 malignant lines having an IC50 value lessthan 1000 compared to the relative insensitivity of normal human celltypes. The majority of human cancer cells had IC50 values that were 2 to3 orders of magnitude lower than most normal human cell types. TABLE 4Summary of Cytotoxicity Assay Results Tumor Type CELL LINE IC₅₀(PFU/well) FIBROSARCOMA HT 1080 2 MELANOMA SKMEL2 8 SKMEL3 2 SKMEL5 4A375 37 MALME-3M 778 HT144 28 BREAST CARCINOMA SKBR3 10 MDA-MB-468 44ZR75-1 78 OVARIAN CARCINOMA SW626 4 PA-1 4 ES-2 13 SKOV-3 24 OVCAR3 34LUNG CARCINOMA H-1299 26 (Large Cell, Low Passage) GLIOBLASTOMA U87MG 25U373MG 765 U138 38 A172 207 BLADDER CARCINOMA HT1197 3 UM-UC-3 54 HT1376422 NEUROBLASTOMA IMR32 41 CERVICAL CARCINOMA HeLa 4 PROSTATE CARCINOMADU-145 31 PC3 3.1 × 10³ COLON CARCINOMA SW620 55 HT29 >1.0 × 10⁶  HEAD-AND-NECK KB 4 CARCINOMA A253 >2.7 × 10³   FaDu >2.9 × 10³  Hep-2 >1.5 × 10⁴   NEUROEPITHELIOMA SK-N-MC 20 SMALL CELL CA, LUNGDMS-114 48 DMS-153 1.1 × 10⁵ NCl-H345 1.2 × 10⁶ SMALL CELL CA, NCl-H6601.0 × 10⁵ PROSTATE LEUKEMIA (AML) K562 5.4 × 10⁴

[0201] TABLE 5 Summary of Cytotoxicity Assay Results Using Normal HumanCells CELL TYPE CELL IC₅₀ (PFU/well) Keratinocytes NHEK 9.0 × 10⁶Fibroblasts CCD-922 1.4 × 10⁵ NHDF 8.1 × 10³ Endothelial Cells HPAEC 5.2× 10⁴ Renal Cells RPTEC 2.7 × 10⁴ Melanocytes NHEM 5.1 × 10⁴ AstrocytesNHA 3.8 × 10³

[0202] TABLE 6 Summary of Cytotoxicity Assay Results Using RapidlyProliferating Normal Human Cells RATE OF PROLIFERATION CELL TYPE IN VIVOIN VITRO IC₅₀ (PFU/well) Bone Marrow Cells Moderate to High 6.2 × 10³CD34+ Enriched to 50% High Breast Epithelial Cells Very Low¹ High¹ 30

EXAMPLE 2

[0203] Use of PPMK107 for the Intratumoral Treatment of Human TumorXenografts (<10 mm and >5 mm) in Athymic Mice

[0204] Athymic mice were injected intradermally with 10 million humantumor cells. After tumors reached a size range from between 5 and 10 mm,a single injection of PPMK107 (at a dose of 3×10⁸ PFU) or saline wasgiven. Almost all tumor types exhibited a rate of complete or partialregression of 50% to 100% (see Table 7) in mice treated with PPMK107.The one exception is the case of the U87MG experiment (experiment I):Although only one of 9 tumors treated with PPMK107 completely regressed,two more virus-treated tumors showed regression of 32% and 20% and twomore virus-treated tumors had slower growth than all 8 tumors treatedwith saline control. Tumor regression was virtually absent in the salinecontrol treated tumors: In all of these experiments (A through I listedin Table 7) only one of 73 control tumors showed regression. Theseresults indicate that diverse tumor types showed responses tointratumoral PPMK107 treatment.

[0205] To examine virus replication within the tumor,immunohistochemical staining for viral antigen (using a monoclonalantibody against the NDV P protein) was performed using the subcutaneousHT1080 fibrosarcoma model. Within 30 minutes of intratumoral injectionof 3×10⁸ PFU of PPMK107, the tumor tissue was negative for viralantigen. However, by day 2 post treatment, intense immunostaining forviral antigen was seen within the tumor, indicating virus replicationwithin the tumor. Importantly, virus replication was specific for thetumor tissue since the neighboring connective tissue and skin wasnegative for viral antigen.

EXAMPLE 3

[0206] Use of PPMK107 for the Intravenous Treatment of Human TumorXenografts (<8.5 mm and >5.5 mm) in Athymic Mice

[0207] Athymic mice were injected intradermally with 10 million humanHT1080 fibrosarcoma cells. After tumors reached a size range frombetween 5 and 8 mm, a intravenous injection(s) of PPMK107 or saline weremade. As shown in Table 8, at the highest virus dose level (1×10⁹ PFU)complete tumor regression was seen in all seven mice. Single injectionsof 3×10⁸ and 6×10⁷ resulted in regression rates of over 90%. While asingle IV injection of 3×10⁸ only a 55% rate of tumor regression, threeIV injections at this dose level yielded a 100% rate of response. Micetreated with IV saline exhibited no evidence of tumor regression. Theseresults indicate that subcutaneous HT1080 tumors are very responsive toIV treatment with PPMK107. TABLE 7 PPMK107 intratumoral treatment ofsubcutaneous human tumor xenografts (<10 mm and <5 mm) in athymic miceComplete Complete + Tumor Tumor Type Expt # Dose N Regression partialRegression HT1080 Fibroscarcoma A 3.00E+08 12 11 11 B 3.00E+08 9 8 8 C3.00E+08 8 8 8 PA-1 Ovarian Carcinoma D 3.00E+08 9 9 9 KB Oral CarcinomaE 3.00E+08 12 7 10 SKMEL5 Melanoma F 3.00E+08 8 5 7 A375 Melanoma G3.00E+08 8 5 7 H 3.00E+08 8 1 4 U87MG Glioblastoma I 3.00E+08 9 1 1

[0208] TABLE 8 PPMK107 intravenous treatment of subcutaneous humanHT1080 fibrosarcoma xenografts (<8.5 mm and <5.5 mm) in athymic miceComplete Complete + % Re- Dose Schedule N Regression partial gression1.00E+09 One Injection 7 7 7 100% 3.00E+08 One Injection 10 9 10 100%6.00E+07 One Injection 11 10 10  91% 2.00E+07 One Injection 11 5 6  55%2.00E+07 Three Injections 7 5 7 100% Every Other Saline One Injection 100 0  0% Saline Three Injections 6 0 0  0% Every Other

EXAMPLE 4

[0209] First Experiment Using PPMK107 for Intratumoral Treatment ofLarge A375 Melanoma Xenografts in Athymic Mice

[0210] Athymic mice were injected intradermally with 10 million A375human melanoma cells. Ten days later, tumors of various sizes weretreated with a single injection PPMK107 (doses of 3×10⁸, 9×10⁸, and1.5×10⁹ PFU) or saline. For those tumor with a single largest dimensionof 10 to 11 mm, all nine completely regressed in response tointratumoral treatment with these doses of PPMK107, while of thosetumors with a single largest dimension of 8 to 9.5 mm, twelve out of 24completely regressed in response to virus therapy (P<0.008; Table 9,section A). No tumor regression was seen in any mouse treated withsaline.

[0211] These same tumors when sorted by tumor volume also indicated ahigh percentage of complete regression in those of larger tumor volume.In response to these doses PPMK107, complete regression occurred in 14out of 17 tumors with volumes >300 mm³ (range of 304 to 397 mm³) and in7 out of 16 tumors with volumes <300 mm³ (range of 144 to 295; P <0.023;Table 9, section B).

[0212] These results indicate that tumors at least 1 cm in length or 300mm³ in volume were at least as sensitive, if not more sensitive, tointratumoral PPMK107 treatment than smaller tumors.

EXAMPLE 5

[0213] Second Experiment Using PPMK107 for Intratumoral Treatment ofLarge A375 Melanoma Xenografts in Athymic Mice

[0214] Tumors were established as in Example 4 ten days after tumor cellinoculation. Treatment consisted of various doses of PPMK107 (3×10⁶ PFU3×10⁷, 3×10⁸, and 1.5×10⁹) or saline, For tumors 10 to 11.5 mm in singlelargest dimension, complete or partial (at least 50%) regressionoccurred in all 28 tumors treated with PPMK107 using these doses incontrast to no regression in any of the saline-treated mice (Table 10,section A).

[0215] When these same tumors were sorted by tumor volume, all 26 tumorsgreater than 300 mm³ (range: 309 to 525 mm³) regressed completely orpartially (at least 50%) in response to PPMK107 in contrast to none ofthe saline treated mice (Table 10, section B).

[0216] These results confirm that tumors at least 1 cm in length or 300mm³ in volume are sensitive to intratumoral PPMK107 treatment. TABLE 9Intratumoral PPMK107 Treatment of Intradermal A375 Melanoma XenograftsTumor Dimension: 8 to 9.5 mm Tumor Dimension: 10 to 11 mm CompleteComplete Treatment Dosage N Regression % N Regression % A. Tumors SortedBased on the Single Largest Dimension PPMK107 1.5 × 10⁹ 8 2 25% 3 3 100%PPMK107 9.0 × 10⁸ 8 7 88% 3 3 100% PPMK107 3.0 × 10⁸ 8 3 38% 3 3 100%Total 24  12  50% 9 9 100% a Saline 6 0  0% 3 0  0% Tumor Volume: <300mm³ Tumor Volume: >300 mm³ Complete Complete Treatment Dosage NRegression % N Regression % B. Tumors Sorted Based on the Tumor VolumePPMK107 1.5 × 10⁹ 6 2 33% 5 3  60% PPMK107 9.0 × 10⁸ 4 3 75% 7 7 100%PPMK107 3.0 × 10⁸ 6 2 33% 5 4  80% Total 16  7 44% 17  14   82% b Saline8 0  0% 1 0  0%

[0217] TABLE 10 Intratumoral PPMK107 Treatment of Intradermal A375Melanoma Xenografts Regressions Complete + Treatment Dose N Complete %Partial % A. Tumors 10 to 11.5 mm (Sorted Based on the Single LargestDimension) 1.5 × 10⁹ 7 7 100% 7 100% 3.0 × 10⁸ 7 6 86% 7 100% 3.0 × 10⁷7 5 71% 7 100% 3.0 × 10⁶ 7 5 71% 7 100% All PPMK107 28 23 82% 28 100%Groups Saline 6 0 0% 0 0% B. Tumors >300 mm³ (Sorted Based on the TumorVolume) 1.5 × 10⁹ 7 7 100% 7 100% 3.0 × 10⁸ 7 6 86% 7 100% 3.0 × 10⁷ 6 467% 6 100% 3.0 × 10⁶ 6 4 67% 6 100% All PPMK107 26 21 81% 26 100% GroupsSaline 5 0 0% 0 0%

EXAMPLE 6

[0218] Third Experiment Using PPMK107 for Intratumoral Treatment ofLarge A375 Melanoma Xenografts in Athymic Mice

[0219] Tumors were established as in Example 4 nineteen days after tumorcell inoculation. Intratumoral treatment consisted of various doses ofPPMK107 (3×10⁸, 3×10⁶, 3×10⁵, 3×10⁴, 3×10³, 3×10² PFU) or saline. Fortumors 12.5 to 14 mm in single largest dimension (volume range: 632 to787 mm³; average volume 698 mm³), tumor regressions of at least 50%occurred in two out of three mice treated with 3×10⁸ PFU in contrast tono regression in both saline-treated mice (Table 11). Using the samedose of PPMK107 (3×10⁸ PFU) to treat tumors with a single largestdimension of 10 to 12 mm (volume range: 320 to 600 mm³; average volume:411 mm³), seven of 8 mice exhibited regression of at least 25% (P<0.001for regression of at least 25% compared to the saline treated mice whichexhibited no regressions, Table 11). Regressions of at least 25% fortumors of length 10 to 12 mm tumors were also seen in mice treated with3×10⁶ PFU, 3×10⁵ PFU, 3×10⁴ PFU, and 3×10³ PFU, but not for mice treatedwith 3×10² PFU or saline (Table 11).

[0220] These results confirm that tumors at least 1 cm in length or 300mm³ in volume are sensitive to intratumoral PPMK107 treatment.

EXAMPLE 7

[0221] Fourth Experiment Using PPMK107 for Intratumoral Treatment ofLarge A375 Melanoma Xenografts in Athymic Mice

[0222] Tumors of largest dimension 10 to 12 mm were established as inExample 4 thirteen days after tumor cell inoculation. Intratumoraltreatment consisted of a single injection of 3×10⁸ PFU of PPMK107 orsaline. Volumes of those tumors treated with PPMK107 ranged from 295 to600 mm³ (average tumor volume of 437 mm³). Groups of mice in eachtreatment group were euthanized on days 0, 2, 3, 4, 7, and 14 for tumorhistology. For those mice observed for a minimum of 4 days, eleven outto 12 mice treated with PPMK107 exhibited regression of at least 25%compared to none of 8 in the saline group (P<0.0001, Table 12). At 2days after PPMK107 treatment, two tumors already exhibited signs ofregression but the degree of regression was less than 25%. TABLE 113^(rd) Experiment Using PPMK107 for the Intratumoral Treatment of A375Melanoma Xenografts (at least 10 mm in size) Regressions Total # of %Treatment N Volume Range Avg Volume Complete Partial^(a) >25% & <50%^(b)Regressions^(c) Regressions^(c) Size: 12.5 to 4 mm 3.0E+08 3 632 to 787698 1 1 0 2 67% Saline 2 717 to 860 788 0 0 0 0 0% Size: 10 to 12 mm3.0E+08 8 320 to 600 411 0 3 4 7 88% 3.0E+06 8 425 to 662 502 0 0 2 225% 3.0E+05 8 245 to 600 421 0 0 1 1 13% 3.0E+04 8 336 to 600 477 0 0 11 13% 3.0E+03 8 281 to 542 349 2 0 0 2 25% 3.0E+02 8 281 to 662 372 0 00 0 0% Saline 8 379 to 666 518 0 0 0 0 0%

[0223] TABLE 12 4^(th) Experiment Using PPMK107 for the IntratumoralTreatment of A375 Melanoma Xenografts (at least 10 mm in size) DayEuthanized Regressions Total # of % Treatment Post Treatment N CompletePartial^(a) >25% & <50%^(b) Regressions^(c) Regressions^(c) Tumor Size:10 to 12 mm 3.0E+08 14 Days 3 0 2 1 3 100% 3.0E+08 7 Days 3 0 2 1 3 100%3.0E+08 4 Days 3 0 2 1 3 100% 3.0E+08 3 Days 3 0 0 2 3 67% 3.0E+08 AllPPMK107 Groups 12 0 6 5 11 92% d,e Saline 14 Days 2 0 0 0 0 0% Saline 7Days 2 0 0 0 0 0% Saline 4 Days 2 0 0 0 0 0% Saline 3 Days 2 0 0 0 0 0%Saline All Saline Groups 8 0 0 0 0 0%

EXAMPLE 8

[0224] Fifth Experiment Using PPMK107 for Intratumoral Treatment ofLarge A375 Melanoma Xenografts in Athymic Mice

[0225] Tumors of largest dimension 10 to 12 mm were established as inExample 4 twenty days after tumor cell inoculation. Intratumoraltreatment consisted of a single injection of 3×10⁸ PFU of PPMK107 orsaline. Volumes of those tumors treated with PPMK107 ranged from 361 to756 mm³ (average tumor volume of 551 mm³). Nine out of 10 mice treatedwith PPMK107 exhibited a regression of at least 25% compared to none of10 in the saline group (P<0.0001, Table 13).

EXAMPLE 9

[0226] First Experiment Using PPMK107 for Intravenous Treatment of LargeHT1080 Fibrosarcoma Xenografts

[0227] Athymic mice were injected subcutaneously with 10 million HT1080human fibrosarcoma cells. Six days later, tumors were treated with asingle injection PPMK107(at a dose of 1.5×10⁹ PFU) or saline. For thosetumors 10 to 11 mm in single largest dimension, five out of six tumorscompletely or partially regressed in response to a single intravenousinjection of PPMK107 compared to none of the saline treated tumors(Table 14, P<0.025). These results indicate that tumors at least 1 cm inlength are sensitive to intravenous PPMK 107 treatment. TABLE 13 5^(th)Experiment Using PV701 for the Intratumoral Treatment of A375 MelanomaXenografts (at least 10 mm in size) Regressions Total # of % Treatment NComplete Partial >25% & <50%^(b) Regressions^(c) Regressions^(c) Size:10 to 12 mm 3.0E+08 10 0 4 5 9 90% d,e Saline 10 0 0 0 0  0%

[0228] TABLE 14 Intravenous Treatment of Subcutaneous HT1080 HumanFibrosarcoma Xenografts in Athymic Mice Complete + Treatment Dose NComplete % Partial % Size: 10 to 11 mm PPMK107 1.5E+09 6 4 67% 5 83% aSaline 4 0 0 0 0

EXAMPLE 10

[0229] Specific Clearing of PPMK107 Infection from Normal but Not TumorCells

[0230] In order to examine the mechanism of tumor-specific killing byNDV strain PPMK107, representative tumor cells were chosen based on thefollowing criteria: a) ability to form tumors as xenografts in athymicmice; b) the tumor xenografts are specifically killed in vivo followingadministration of PPMK107; c) the tumors cells exhibit killing byPPMK107 in vitro at virus concentrations that are several logs below theconcentration to kill resistant, normal cells; and d) tumor cells mustbe easily distinguished from the normal cells when present as aco-culture. Xenograft tumors comprised of KB head and neck carcinomacells exhibit 83% complete or partial regression in response to a singleintratumoral injection of PPMK107, are more than four logs moresensitive to killing by PPMK107 in vitro than are normal primary skinfibroblasts (CCD922-sk), and are easily distinguished from CCD922-skcells when present as a co-culture.

[0231] Accordingly, co-cultures of KB and CCD922-sk cells were infectedat a multiplicity of infection (m.o.i., the ratio of virus added percell) of 0.0005 and the course of the infection followed for 5 days byimmunohistochemical staining for a viral antigen (NDV P protein).Infection of normal cells peaked at 2 days with little or no apparentcell death as determined by visual inspection of the cell monolayer. Onthe third day post-infection the amount of viral expression in thenormal cells decreased significantly, while infection of the tumor cellswas clearly apparent. The amount of viral antigen virtually disappearedin the normal cells on days 4 and 5, while the infection in the tumorcells progressed rapidly through the tumor cell population resulting indestruction of the majority of the tumor cells present in theco-culture.

[0232] Thus, normal cells were infected and easily cleared the infectionin a manner consistent with the anti-viral effects of IFN. The tumorcells were unable to establish an anti-viral state in response and werekilled by the unabated viral growth, despite the presence ofphysiologically effective concentrations of IFN secreted into the mediaby the normal cells.

EXAMPLE 11

[0233] Demonstration that Interferon is an Important Component of ViralClearing in Normal CCD922-sk Cells

[0234] The hypothesis that interferon was mediating the ability ofCCD922-sk cells to clear the infection of PPMK107 was tested. Polyclonalneutralizing antibodies to human interferon-a or human interferon-β,used alone or in combination, were added daily to cultures of CCD922-skcells infected with PPMK107 at an moi of 0.0005 and the progress of theinfection followed for three days. The amount of viral antigen presentin the cells increased in proportion to the concentration ofneutralizing antibody, with the effect of the anti-interferon-β antibodybeing more marked than that of the anti-interferon-α antibody;consistent with reports that fibroblasts produce predominantly the betaform of interferon.

[0235] The ability to make the normally insensitive cells moresusceptible to infection with PPMK107 through the addition ofneutralizing antibody to interferon supports the hypothesis that a keydifference between the sensitivity of normal and tumor cells to killingby PPMK107 lies in the ability of normal cells, but not tumor cells, toestablish an interferon-mediated anti-viral response.

EXAMPLE 12

[0236] Demonstration that Interferon-β is an Important Component ofViral Clearing in Other Normal Cells

[0237] In this experiment, it was determined that another normal cell(NHEK, normal human epithelial cells) known to be quite resistant tokilling by PPMK107, was made more sensitive through the addition ofpolyclonal anti-interferon-β antibody to a culture of infected cells.NHEK (normal human epithelial keratinocyte) cells were infected at anmoi of either 0.0005 or 0.05 and had antibody added daily over fivedays.

[0238] In the cultures infected at the low moi (0.0005), antibodydependent augmentation of viral antigen expression was clear at fivedays post-infection, but was less clear earlier in the experiment.Antibody addition to cultures infected with PPMK107 at an moi of 0.05resulted in a marked increase in viral antigen at 4 and 5 dayspost-infection. At 2 and 3 days post-infection the addition ofneutralizing antibody resulted in less accumulation of viral antigen(FIG. 1).

[0239] The culture supernatants from the high moi samples were alsotitrated for the amount of infectious virus present by plaque assay onhuman HT1080 fibrosarcoma tumor cells; the standard assay system in ourlaboratory. Results from this analysis demonstrated that at five dayspost-infection there was 19-fold increase in the amount of infectiousvirus in the antibody-treated cultures relative to mock-treated controls(FIG. 1).

[0240] These results suggest a general mechanism by which normal cellsare protected from killing by PPMK107 through an interferon-relatedmechanism.

EXAMPLE 13

[0241] Comparison of the Effect of Interferon-β on PPMK107 Infection inTumor and Normal Cells

[0242] A comparison of the effect of exogenously added interferon-β onthe infection of normal (CCD922-sk) and tumor cells of high (KB) orintermediate (HEp2) sensitivity PPMK107 was performed. Separate culturesof the three cells were treated with interferon-β at 20, 200, or 2000units/ml 1 day pre- and 2 days post-infection at an moi of 0.0005.

[0243] At 3 days post-infection the low level of viral antigenexpression present in the normal cells was eliminated at all doses ofinterferon used. Conversely, the addition of interferon to the highlysensitive KB tumor cells at concentrations of 2 or 200 units/mldecreased relative levels of viral antigen expression 2-fold, withcomplete suppression at 1000 units/ml interferon. The intermediatelysensitive HEp-2 cells responded to the exogenous interferon by clearingviral antigen expression at all of the interferon doses used (FIG. 2).

[0244] The pattern of sensitivity in the KB and CCD922-sk cells to theanti-viral effects of exogenously added interferon-β was inverselyproportional to the sensitivity of these cells to killing by PPMK107.The ability of the HEp-2 cells to respond to the effects of interferonindicates that these cells are able to efficiently utilize theconcentrations of interferon used in this experiment. Similarly, theresponse of the KB cells to the high doses of interferon suggests thatthe inability to establish an interferon-mediated anti-viral responsedoes not result from an absolute defect in the interferon pathway, butrather a relative insensitivity compared to normal cells.

EXAMPLE 14

[0245] Effect of Low Concentrations of Interferon-β on the Infection ofNormal and Tumor Cells by PPMK107

[0246] In this experiment normal (CCD922-sk) and tumor (KB) cells weretreated with low concentrations of interferon-β (0.2, 2, and 20units/ml) 1 day before and 2 days post-infection with PPMK107 at an moiof 0.05.

[0247] Under these conditions the normal cells experienced adose-dependent decrease in the amount of viral antigen, while therelative levels of viral antigen in the tumor cells was unaffected bythe addition of exogenous interferon (FIG. 3).

EXAMPLE 15

[0248] PPMK107 Purification

[0249] Method A

[0250] PPMK107 was derived from the mesogenic Newcastle disease virusstrain Mass-MK107 by triple plaque purification. Approximately 1000 PFUs(plaque forming units) of live PPMK107 were inoculated into theallantoic fluid cavity of each 10 day old embryonated chicken egg. Afterincubation at 36° C. for 46 hours, the eggs were chilled and then theallantoic fluid was harvested. Cells and cell debris were removed fromthe allantoic fluid by centrifugation at 1750×g for 30 minutes. Theclarified allantoic fluid (supernatant containing virus) was thenlayered over a 20%/55% discontinuous sucrose gradient) and centrifugedat approximately 100,000×g for 30 minutes. The purified virus washarvested from the 20%/55% interface and dialyzed against saline toremove the sucrose.

[0251] Method B

[0252] In another advantageous embodiment, the clarified allantoic fluidwas frozen at −70° C. After thawing, the fluid was maintained at 1 to 4C overnight and then the contaminating material was removed from thevirus suspension by means of centrifugation (1750×g for 30 minutes).This material was further processed using the discontinuous sucrosegradient on the ultracentrifuge as above.

[0253] Method C

[0254] In another advantageous embodiment, ultracentrifugation on thediscontinuous sucrose gradient was accomplished by means of a continuousflow ultracentrifuge.

[0255] Method D

[0256] In another advantageous embodiment, harvested allantoic fluid isdiluted with a buffer containing 5% mannitol and 1.0% 1-lysine, pH 8.0(ML buffer) and is clarified and exchanged with ML buffer by tangentialflow filtration (TFF) through filters with a nominal pore size of 0.45μ.The permeate containing the clarified virus in ML buffer is collectedand virus is purified by TFF through filters with a nominal cut-off of300,000 daltons in ML buffer. The concentrated, purified virus in MLbuffer is collected as the retentate from this step and is again dilutedwith ML buffer before being applied to a Sephacryl S500 (Pharmacia) gelpermeation column equilibrated with ML buffer. Fractions containingpurified virus are collected, pooled and can be reconcentrated by TFFthrough filters with a nominal cut-off of 300,000 daltons with MLbuffer.

[0257] Results

[0258] *Clonal Virus

[0259] After generation of PPMK107 by plaque purification, eightindividual molecular clones from the population of virions were found tohave an identical sequence (e.g. a homology of 100%) of over 300contiguous nucleotides within the fusion protein gene of NDV. PPMK107 isa clonal virus with a high degree of genetic homogeneity.

[0260] PFU/mg Protein

[0261] One quantitative means of measuring purity is by determination ofa PFU/mg protein. Higher values indicate a greater level of purity.Using Method A, PFU/mg values of at least 4.8×10¹⁰ were achieved (seeTable 15). Using Method C, PFU/mg protein values of at least 2.0×10¹⁰were achieved. For a mesogenic strain of NDV, a literature value forthis measurement of purity has not been found. The best estimate for amesogenic strain of NDV is the virus preparation (NDV Mass/MK107, lotRU2, prepared as in Faaberg K S and Peeples, M D, 1988, J. Virol.62:586; and Bratt, M A and Rubin, H. 1967, Virology 33:598-608). ThisRU2 lot was found to have a PFU/mg of 1.3×10⁹ PFU/mg of protein. Thepurity values achieved by Method A are approximately 40 times betterthan what the Peeples method achieved (see Table 15).

[0262] *Particle per PFU Ratio

[0263] Another quantitative means of measuring purity is bydetermination of a ratio of particles per PFU. Lower values indicate agreater level of purity. Particle counts were done by electronmicroscopy using standard methods. Using either Method A or Method B,particles per PFU values near one were achieved (Table 15). TABLE 15Virus Purity Virus Preparation PFU per Particle Method Virus Lot # mgprotein per PFU Preferred Method A PPMK107 L2 4.8 × 10¹⁰ 0.80 L4 6.9 ×10¹⁰ NT^(a) L5 6.6 × 10¹⁰ NT L6 7.7 × 10¹⁰ 0.55 L7 6.1 × 10¹⁰ NTPreferred Method C PPMK107 D004 2.0 × 10¹⁰ 0.32 D005 4.5 × 10¹⁰ 0.52D010 4.4 × 10¹⁰ NT Preferred Method D PPMK107 RD2 5.6 × 10¹⁰ NT RD3 5.0× 10¹⁰ NT

[0264] Virus preparations using Methods A and C also permittedpurification of NDV to a level substantially free of contaminating eggproteins. For the PPMK107 lot 7 preparation using Method A, ovalbumin,was not detectable in a Western blot using (1) 1.7×10⁹ PFU of purifiedvirus per well (3.3 cm in width) run on an SDS-PAGE (sodium dodecyl 10sulfate-polyacrylamide gel electrophoresis) gel (1 mm thick); (2) anitrocellulose membrane for transfer; and (3 rabbit anti-ovalbumin(Cappel rabbit IgG fraction at a 1:200 dilution of a 4 mg/ml antibodyconcentration). For PPMK107 preparations using Method D and analyzed bySDS-PAGE followed by silver staining, no band corresponding to ovalbuminwas observed.

EXAMPLE 16

[0265] Use of PPMK107 To Prevent Deaths from ES-2 Ovarian CarcinomaAscites in Athymic Mice

[0266] In this experiment, all of the athymic mice (female, NCR nu/nu, 8weeks old) were given an intraperitoneal injection of 10⁶ ES-2 cells.Seven days later before ascites had developed, they were treatedintraperitoneally with saline or PPMK107 (at 1×10⁹ PFU). As shown inFIG. 4, there was a markedly improved survival in the animals treatedwith PPMK107 compared to saline. The majority of the mice in the salinetreated group had developed ascites by seven days post-treatment and byday 38, all of these animals had died. In marked contrast, 92% of themice treated with PPMK107 were still alive by day 38 and 25% of theseanimals were long term survivors (>120 day survival).

EXAMPLE 17

[0267] PPMK107 Treatment of ES-2 Ovarian Carcinoma in Athymic Mice WhenAscites is Present

[0268] In this experiment, all of the athymic mice (female, NCR nu/nu, 8weeks old) were given an intraperitoneal injection of 10⁶ ES-2 cells.Fourteen days later when the majority of mice had developed ascites, themice without ascites were excluded and the mice with ascites wererandomized into 7 intraperitoneal treatment groups (PPMK107-onetreatment on day 0; PPMK107-two treatments for the first week;PPMK107-one treatment each week; PPMK107-two treatments each week;saline-one treatment on day 0; saline-two treatments for the first week;saline-two treatments each week). A dose of 1×10⁹ PFU/mouse was used foreach virus treatment. All of the mice before the first treatment and anyadditional treatments were drained of the ascites fluid. Day 0 refers tothe day of first treatment.

[0269] The degree of ascites for each mouse was quantified and noted asfollows: Ascites Score Degree of Ascites 1.0 Animal appearsnormal-little or no ascites present 2.0 Abdomen slightly distended;animal is capable of normal functions 3.0 Abdomen distended; animal isslow-moving, hunched with a staggered gait. 4.0 Abdomen completelydistended; animal moribund 5.0 Death after ascites development

[0270] As shown in Table 16, all of the saline-treated animals had moreadvanced ascites than the PPMK107-treated animals on both days 7 and 10.On day 7 post initial treatment, each the saline group had averageascites scores above 3.5 while all of the PPMK107-treated groups hadaverage ascites scores at 3.0 or below. Similarly on day 10 post initialtreatment, each the saline group had average ascites scores above 4.5while all of the PPMK107-treated groups had average ascites scores at4.1 or below. These results indicate that ascites fluid production wasmarkedly decreased in virus-treated animals compared to saline controls.TABLE 16 PPMK107 Treatment of ES-2 Ovarian Carcinoma in Athymic MiceWhen Ascites is Present. Average Average Ascites # of Ascites Score,Score, Treatment Mice Day 7 Day 10 Saline x1 12 4.3 4.7 Saline x2 12 3.74.6 Saline x2 each wk 12 4.3 4.8 PPMK107 x1 17 3.0 4.1 PPMK107 x2 17 2.33.6 PPMK107 x1 each wk 17 2.6 2.6 PPMK107 x2 each wk 17 2.2 3.6

EXAMPLE 18

[0271] Use of a Desensitizing Dose of PPMK107 to Reduce the Lethality ofa Subsequent Dose of PPMK107

[0272] C57BL/6 mice (seven weeks old) were injected intravenously on day0 with either saline or a desensitizing dose of PPMK107 (3×10⁸PFU/mouse). Two days later each set of mice were further subdivided intogroups for intravenous dosing with saline or PPMK107 (at doses of Ix10⁹, 2.5×10⁹, 5×10⁹, and 1×10¹⁰ PFU/mouse). As shown in Table 17, whensaline was used to pretreat the mice, deaths were recorded in the micesubsequently dosed with 2.5×10⁹, 5×10⁹, and 1×10¹⁰ PFU. The doses of5×10⁹ and 1×10¹⁰ PFU were 100% lethal to the mice pretreated withsaline. In contrast, no deaths were seen in any group of mice given adesensitizing dose of PPMK107 on day 0 followed by PPMK107 injection twodays later at dose levels up to 1×10¹⁰ PFU. These data indicate thatPPMK107 can be used to prevent the lethality of subsequent dosing withthis same agent. Furthermore, the maximal tolerated dose of PPMK107 canbe raised by an approximate order of magnitude when using this virus asa desensitizing agent. TABLE 17 Use of a Desensitizing Dose of PPMK107to Reduce the Lethality of a Subsequent Dose of PPMK107. Dose on # of #of % Group Injection on Day 0 Day 2 Mice Deaths Lethality 1 SalineSaline 8 0 0 2 Saline PPMK107, 8 0 0 1.0E+09 3 Saline PPMK107, 8 3 382.5E+09 4 Saline PPMK107, 8 8 100 5.0E+09 S Saline PPMK107, 8 8 1001.0E+10 6 PPMK107, 3E+08 Saline 8 0 0 7 PPMK107, 3E+08 PPMK107, 8 0 01.0E+09 8 PPMK107, 3E+08 PPMK107, 8 0 0 2.5E+09 9 PPMK107, 3E+08PPMK107, 8 0 0 5.0E+09 10  PPMK107, 3E+08 PPMK107, 8 0 0 1.0E+10

EXAMPLE 19

[0273] Slower Intravenous Injection Rate Reduces the Toxicity of PPMK107

[0274] Twenty two athymic mice (8 weeks old) were anesthetized with acombination of ketamine/xylazine and placed into a restrainer to helpinhibit their movement during the injection process to allow for eithera slow or rapid injection of PPMK107. For the slow injection group, 0.2mL of 4×10⁹ PFU of PPMK107 in saline was injected intravenously over a 4minute period with 0.01 mL given every 10 to 15 seconds. The rapidinjection group received the same dose and volume but over a 30 secondperiod. As shown in Table 18, the animals receiving their dose ofPPMK107 over 4 minutes had half as much maximal weight loss (recorded onday 2 after dosing) as the animals receiving the same IV dose over 30seconds. These results indicate that PPMK107 has less toxicity and issafer for intravenous administration when injected at such slower rates.TABLE 18 Slower IV Injection of PPMK107 Results in Reduced Toxicity.Length of Time That Dose was # of Maximal Percent Group AdministeredMice Weight Loss Rapid Injection of 30 seconds 11 12% 4E+09 SlowInjection of  4 minutes 11  6% 4E+09

EXAMPLE 20

[0275] Use of PPMK107 in the Treatment of Patients with Advanced Cancer.

[0276] PPMK107 has been tested in a phase 1 clinical trial in the U.S.A.by the intravenous route. Twenty-three patients with advanced solidtumors, no longer amenable to established therapies, have been treatedwith PPMK107. Seventeen of these patients have received a single dosefor the initial treatment course. Six other patients are receiving threedoses per week for one week for the initial treatment course. The sizesof each patient's tumors were followed once per month. Patients with atleast stable disease (less than 25% increase and less than 50% decreasein the sum of the products of all measurable tumors in the absence ofany new lesions) were eligible for additional treatment courses eachmonth.

[0277] Regression of a Palpable Tumor

[0278] A 68 year old female with colon carcinoma had a palpableabdominal tumor among her widespread metastases. After a single IVtreatment with PPMK107, this patient experienced a 91% regression ofthis single abdominal wall tumor over the course of two weeks (Table19). Measurements of the tumor one day after dosing (3.75×3 cm) weresimilar to the baseline measurements of 4×3 cm. However, by day 7 postdosing, the tumor had decreased in size to 2×2 cm and continued todecrease in size to 1.5×1.5 cm by day 14 after PPMK107 dosing. Previousto PPMK107 treatment, this tumor mass had been rapidly growing with a1065% increase in tumor volume in the two weeks before PPMK107 dosing.This patient went off study because of increased growth of the tumorelsewhere. TABLE 19 Size of Palpable Abdominal Wall Tumor in Patient#123 (68 year old Female with Metastatic Colon Carcinoma) After a SingleIV PPMK107 Dose of 12 Billion PFU/m². % Tumor Tumor Volume ReductionTime After Dimensions (0.5 × L × in Tumor Date Dosing (L × W, cm³) W ×W, cm³) Volume Jul. 23, 1998 Day 0 4 × 3 18. — Jul. 24, 1998 Day 1 3.75× 3   16.9  6% Jul. 30, 1998 Day 7 2 × 2 4.0 78% Aug. 6, 1998  Day 141.5 × 1.5 1.7 91%

[0279] Stabilization of Cancer

[0280] Eight other patients, all of whom previously had tumorprogression with conventional cancer therapies, experienced benefit inthe form of stabilization of their advanced cancer after PPMK107 dosing.These patients with stable disease represent those with diverse types ofcancer including renal cancer, pancreatic cancer, breast cancer and lungcancer. After three months of PPMK107 treatment, a 67 year old man withadvanced and widely metastatic renal cancer currently had stable diseasewith no indications of any new growth and no indication of an increasein tumor size. There has been a higher rate for stable disease benefitwith higher doses of PPMK107: Two out of 6 patients with stable disease(33% of patients) at the first two single dose levels (5.9 and 12billion PFU per m²) and 4 out of 5 patients (80% of patients) withstable disease at the highest single dose level (24 billion PFU per m²(Table 20). TABLE 20 Treatment of Patients with Advanced Cancer withPPMK107 # of Dose Patients # of % of Level Treated Patients PatientsTypes of Cancer with Stable (Billion at this with with Disease for atLeast One PFU per Dose Stable Stable Month & Length of Stable in²) LevelDisease Disease Disease   5.9 6 2 33% Renal Cancer- Ongoing 3 monthsLung Cancer- Ongoing 2 months 12 6 2 33% Pancreatic Cancer- Ongoing 2months Ovarian Cancer- Ongoing 1 month 24 5 4 80% Breast Cancer- Ongoing1 month Breast Cancer- Ongoing month Lung Cancer- Ongoing 1 monthPancreatic Cancer- Ongoing 1 month Total 17  8 47% Noted Above.

[0281] Reduction in Pain Medication

[0282] One patient at the single dose 5.9 billion PFU/m² dose levelbenefited from PPMK107 treatment in the form of symptomatic relief ofcancer pain as denoted by a reduction in narcotic pain medication.

[0283] Desensitization

[0284] A clear desensitizing effect from the first dose (at 5.9 billionPFU/m2) is seen on subsequent doses (also at 5.9 billion PFU/m²) withinthe same week. In general, the reported side effects from second andthird doses have been much less. For example, the first 4 patients inthis multidose treatment regimen (three doses per week for one week) hadfever after the first dose in spite of receiving prophylacticantipyretic treatment with acetaminophen and ibuprofen. The majority ofthese patients had no fever after receiving the second and third doses,even in cases in which they did not receive antipyretics. This indicatesthat administration of the first dose in the three times per weekschedule reduces the toxicity for the second and third doses.

[0285] Dosing Through Neutralizing Antibodies in Serum

[0286] Using the dose range in this phase I study (≧5.9 billion PFU/m²),there is also clear indication that one can effectively deliver virus topatients even if they have generated neutralizing antibodies. A 72 yearold woman with pancreatic cancer at the 12 billion PFU/m² single doselevel has had stable disease for 2 months since beginning PPMK107treatment. A second course (consisting of a single IV dose of PPMK107)was administered one month after the first dose when the patient hadproduced neutralizing antibodies in her serum. Seven days after thissecond course, her urine was positive for PPMK107 at a titer of at least40 PFU per mL. This result indicates that the neutralizing antibodies toPPMK107 in this patient's serum was not able to completely inhibit thevirus with a second treatment course.

EXAMPLE 21

[0287] Summary of Cytotoxicity Assay Results with Newcastle DiseaseVirus PPNJROAKIN

[0288] Human tumor cells and normal cells were grown to approximately80% confluence in 24 well tissue culture dishes. Growth medium wasremoved and PPNJROAKIN, a plaque purified clone of the mesogenicNewcastle disease virus strain New Jersey Roakin-1946, was added in 10fold dilutions ranging from 10⁷ plaque forming units (PFU)/well to 1PFU/well. Controls wells with no virus added were included on eachplate. Virus was adsorbed for 90 minutes on a rocking platform at 37° C.At the end of the incubation period, the viral dilutions were removedand replaced by 1 ml of growth medium. Plates were then incubated for 5days at 37° C. in 5% CO2. Cytotoxicity was quantified by using acalorimetric MTT (2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) assay (Cell Titer 96, catalog #G4000, Promega Corporation,Madison Wis. 53711) monitored at 570 nm, that detects mitochondrialenzyme activity (Mosman, T., 1983, J. Immunol. Methods 65:55). Theviability in the virus treated wells was expressed as a percent of theactivity in untreated control wells. The data was plotted graphically asPFU/well vs. viability as a percent of control. The IC5O was calculatedas the amount of virus in PFU/well causing a 50% reduction in the amountof viable cells. TABLE 21 Summary of Cytotoxicity Assay Results withPPNJROAKIN. IC₅₀ Cell Type Cell Line (PFU/well) Fibrosarcoma HT1080 13.8Head and Neck KB  2.4 Carcinoma Normal Fibroblast CCD922sk 1.2 × 10⁴

[0289] These results (Table 21) show that PPNJROAKIN demonstratestumor-selective killing of at least two different human tumor cells(HT1080 and KB) relative to normal skin fibroblasts. The IC50 values forthe two tumor cell lines are between 800 and 5000-fold lower than thatfor normal cells.

EXAMPLE 22

[0290] Summary of Cytotoxicity Assay Results with Newcastle DiseaseVirus PPCONN70726

[0291] Human tumor cells and normal cells were grown to approximately80% confluence in 24 well tissue culture dishes. Growth medium wasremoved and PPCONN70726, a plaque purified clone of the mesogenicNewcastle disease virus strain Connecticut 70726-1946, was added in 10fold dilutions ranging from 10⁷ plaque forming units (PFU)/well to 1PFU/well. Controls wells with no virus added were included on eachplate. Virus was adsorbed for 90 minutes on a rocking platform at 37° C.At the end of the incubation period, the viral dilutions were removedand replaced by 1 ml of growth medium. Plates were then incubated for 5days at 37° C. in 5% C02. Cytotoxicity was quantified by using acolorimetric MTT (2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) assay (Cell Titer 96, catalog #G4000, Promega Corporation,Madison Wis. 53711) monitored at 570 nm, that detects mitochondrialenzyme activity (Mosman, T., 1983, J. Immunol. Methods 65:55). Theviability in the virus treated wells was expressed as a percent of theactivity in untreated control wells. The data was plotted graphically asPFU/well vs. viability as a percent of control. The IC50 was calculatedas the amount of virus in PFU/weIl causing a 50% reduction in the amountof viable cells. TABLE 22 Summary of Cytotoxicity Assay Results withPPCONN70726. Cell Type Cell Line IC₅₀ (PFU/well) Head and Neck KB 18.1Carcinoma Glioblastoma U87MG  12.7 Glioblastoma U373MG 879   NormalFibroblast U373MG 7.3 × 10⁴

[0292] These results (Table 22) show that PPCONN70726 demonstratestumor-selective killing of at least three different human tumor cells(KB, U87MG, and U373MG) relative to normal skin fibroblasts. The IC50values for the two tumor cell lines are between 80 and 5000-fold lowerthan that for normal cells.

EXAMPLE 23

[0293] Intratumoral Treatment of HT1080 Fibrosarcoma Xenografts inAthymic Mice Using PPMK107, PPNJROAKIN, or PPCONN70726

[0294] In this experiment, athymic mice (female, NCR nu/nu, 5 to 6 weeksold) received a subcutaneous injection of 10⁷ HT1080 tumor cells. Fourdays later when tumors reached a size range of 6 to 8.5 mm, mice weretreated intratumorally with saline, PPMK107 (at 1×10⁸ PFU), PPNJROAK1N(at 1×10⁸ PFU), or PPCONN70726 (at 1×10⁸ PFU). As shown in Table 23,tumor regression was noted in mice treated with these three viruses(PPMK107, PPNJROAKIN, and PPCONN70726). After PPMK107 treatment of 12mice, four experienced complete tumor regression and six experiencedpartial regression. After PPNJROAKIN treatment of 12 mice, one mouseexperienced complete tumor regression and two experienced partialregression. After PPCONN70726 treatment of 12 mice, three experiencedcomplete tumor regression and two experienced partial regression. Notumor regression was noted in any of the animals treated with saline.These results show that the three mesogenic strains of NDV can causetumor regression. TABLE 23 Regression of HT1080 Fibrosarcoma Tumors inAthymic Mice after Treatment with One of Three Viruses (PPMK107,PPNJROAKIN and PPCONN70726) Each at a Dose of 1 × 10⁸ PFU. It ofRegression Treatment Mice Partial (PR) Complete (CR) PR + CR (%) PPMK10712 6 4 10 (83%)  PPNJROAKTN 12 2 1 3 (25%) PPCONN70726 12 2 3 5 (42%)Saline 11 0 0 0 (0%) 

EXAMPLE 24

[0295] Effects of PPMK107, PPNJROAKIN, PPCONN70726 After IntracerebralInjection in Immunodeficient Athymic (nu/nu) and ImmunocompetentHeterozygote (nu/+) Mice

[0296] Fifty-six athymic mice (nu/nu) and 56 immunocompetentheteroxygote (nu/+) mice were given stereotaxic intracerebral injectionswith either saline, PPMK107, PPNJROAKIN, or PPCONN70726. Eightadditional mice of each type were used as untreated controls. Viruseswere used at one of two dose levels (2×10⁴ or 3.5×10⁶ PFU/mouse). Asshown in Table 24, all of the heterozygote nu/+ mice treated with eachof the three viruses at the two dose levels survived through day 39 withthe exception of one mouse at the lower PPCONN70726 dose level that waseuthanized for non-neurological symptoms. Athymic nu/nu animals treatedwith either PPMK107 or PPCONN70726 had significantly less survival thanthe heterozygotes. This was especially true for the highest PPMK107 orPPCONN70726 virus dose of 3.5×10⁶ PFU/mouse where only 13% (1 of 8) ofthe athymic animals in each virus group survived through day 39. Incontrast, there was 75% survival of the PPNJROAKIN-treated athymic miceat this same dose level (3.5×10⁶ PFU/mouse). These data indicate thatPPNJROAKIN is better tolerated in the brains of athymic mice than theother two virus strains. TABLE 24 Survival of Mice FollowingIntracerebral Injection of PPMK107, PPCONN70726, and PPNJROAKIN %Survival Intracranial Injection # of Mice at Day 39 nu/+ Untreated 8 100nu/+ Saline 8 100 nu/+ PPMK107, 2E+04 8 100 nu/+ PPMK107, 3.5E+06 8 100nu/+ PPCONN70726, 2E+04 8    88 * nu/+ PPCONN70726, 3.5E+06 8 100 nu/+PPNJROAKIN, 2E+04 8 100 nu/+ PPNJROAKIN, 3.5E+06 8 100 nu/nu Untreated 8100 nu/nu Saline 8 100 nu/nu PPMK107, 2E+04 8  75 nu/nu PPMK107, 3.5E+068  13 nu/nu PPCONN70726, 2E+04 8  75 nu/nu PPCONN70726, 3.5E+06 8  13nu/nu PPNJROAKiN, 2E+04 8 100 nu/nu PPNJROAKIN, 3.5E+06 8  75

EXAMPLE 25

[0297] Summary of Cytotoxicity Assay Results with Sindbis VirusPPSINDBIS-Ar339

[0298] Human tumor cells and normal cells were grown to approximately80% confluence in 24 well tissue culture dishes. Growth medium wasremoved and PPSINDBIS-Ar339, a plaque purified clone of Sindbis Ar-339was added in 10 fold dilutions ranging from 10⁷ plaque forming units(PFU)/well to 1 PFU/well. Controls wells with no virus added wereincluded on each plate. Virus was adsorbed for 90 minutes on a rockingplatform at 37° C. At the end of the incubation period, the viraldilutions were removed and replaced by 1 ml of growth medium. Plateswere then incubated for 5 days at 37° C. in 5% C02. Cytotoxicity wasquantified by using a colorimetric MTT(2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay(Cell Titer 96, catalog #G4000, Promega Corporation, Madison Wis. 53711)monitored at 570 nm, that detects mitochondrial enzyme activity (Mosman,T., 1983, J. Immunol. Methods 65:55). The viability in the virus treatedwells was expressed as a percent of the activity in untreated controlwells. The data was plotted graphically as PFU/well vs. viability as apercent of control. The IC50 was calculated as the amount of virus inPFU/well causing a 50% reduction in the amount of viable cells. TABLE 25Summary of Cytotoxicity Assay Results with PPSINDBIS-Ar339 Cell TypeCell Line IC₅₀ (PFU/well) Pancreatic Panc-1* 69 Carcinoma ColorectalSW620* 13 Carcinoma Colorectal SW1463 1.8 × 10⁵ Carcinoma Non-small cellLung A427  >1 × 10⁶  carcinoma Non-small cell Lung A549 5.2 × 10⁴carcinoma Renal carcinoma A498 2.4 × 10⁴ Renal carcinoma Caki-1 3.4 ×10⁴ Fibrosarcoma HT1080 7.4 × 10⁵ Normal Keratinocyte NHEK 2.0 × 10⁵Normal Fibroblast CCD922sk 1.6 × 10⁵

[0299] The cellular receptor for Sindbis virus on mammalian cells is thehigh affinity laminin receptor, that is expressed mainly on cells ofepithelial lineage, but is often overexpressed in many metastatic cancercells like the Panc-1 pancreatic carcinoma line, and the SW620 colonadenonocarcinoma cell line (Campo et al., (1992) Am. J. Pathol. 141,1073-1083; Yow et al., (1988) Proc. Natl Acad Sci. 85, 6394-6398). Incontrast, the rectum adenocarcinoma cell line SW1423 is known to expressvery low levels of high affinity laminin receptor mRNA (Yow et al.,(1988) Proc. Natl Acad Sci, 85, 6394-6398), and is more than 4 order ofmagnitude more resistant to killing by PPSINDBIS-Ar339 than SW620 cells.These results (Table 25) demonstrate that cells that are tumorigenic andexpress high levels of the high affinity laminin receptor are moresensitive to killing by Sindbis Clone PPSINDBIS-Ar339 than other tumoror normal cells.

EXAMPLE 26

[0300] VSV Killing of Tumorigenic and Non-tumorigenic Cells in thePresence of Interferon

[0301] In 96 well plates, tumorigenic KB and HT1080 cells (3×10⁴ cellsper well) and non-tumorigenic WISH cells (2.5×10⁴ cells per well) wereseeded in the presence of serially diluted interferon-α ranging from2800 to 22 Units/ml and allowed to incubate for 24 hours at 37° C. Thecells were then infected with vesicular stomatitis virus (VSV, Indianastrain) at an moi of 10. Controls were included for cells withoutinterferon, and cells without interferon or virus. The cells wereincubated at 37° C. for 24 hours. Cytotoxicity was quantified by using acolorimetric MTT (2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) assay (Cell Titer 96, catalog #G4000, Promega Corporation,Madison Wis. 53711) monitored at 570 nm, that detects mitochondrialenzyme activity (Mosman, T., 1983, J. Immunol. Methods 65:5 5). Theviability in the virus treated wells was expressed as a percent of theactivity in control wells not receiving virus. TABLE 26 Comparison ofthe Cell Killing Activity of VSV in Cells Treated with ExogenousInterferon. Percent Viable Cells WISH HT1080 KB  100 U/ml IFN 50  6  01000 U/ml IFN 95 20 12

[0302] These results (Table 26) demonstrate that VSV is able toselectively kill tumor cells deficient in interferon responsiveness (seeExample 27). WISH cells (human amnion cells) are a well established cellline for the use in interferon bioassays because of their ability torespond efficiently to interferons.

EXAMPLE 27

[0303] Interferon Responsiveness in Cells Sensitive or Resistant toKilling by PPMK107

[0304] Individual cell lines were grown to near confluence in 96 wellmicrotiter plates and treated with between 5 and 5000 U/ml of IFNαA for24 hours. The cultures were then infected with PPMK107 at an moi of 1.0and cultured for an additional 24 hours. Following chemical fixation,the amount of viral expression was quantified by immunohistochemistryusing a soluble indicator dye. The amount of virus growth is representedas the percent of P antigen expressed relative to control cellsuntreated with interferon (FIG. 5). In this assay, interferon responsivecells manifest at least a 50% decrease in the viral antigen in responseto interferon. Cells in FIG. 5 that are sensitive to PPMK107 areindicated by the solid lines; cells less sensitive are indicated by thedashed lines.

[0305] The results of this experiment show a strong correlation betweenthe resistance of the cell line to the antiviral effects of exogenousinterferon and the relative sensitivity of the cell to killing byPPMK107 (indicated by the IC50 value shown in parentheses next to thecell line name in the graph legend, see FIG. 5). For example, followingpretreatment with 5 U/ml of interferon, 6 of 7 (86%) cell linesnonresponsive to interferon are sensitive to killing by PPMK107; whenpretreated with 500 U/ml of interferon, all (4 of 4) of thenonresponsive cell lines are sensitive to killing by PPMK107.

[0306] The data above also present an example of a screening assay toidentify candidate cells that are likely to be sensitive to killing byPPMK107 or other interferon-sensitive viruses. For example, infectedcells expressing significant (e.g., more than 50% of controls) viralantigen following pretreatment with exogenous interferon would beconsidered interferon deficient and thereby sensitive to viraloncolysis.

[0307] The foregoing is intended as illustrative of the presentinvention but not limiting. Numerous variations and modifications may beeffected without departing from the true spirit and scope of theinvention.

What is claimed is:
 1. A method of infecting a neoplasm in a mammal witha virus comprising administering an interferon-sensitive,replication-competent clonal RNA virus to said mammal.
 2. A method ofinfecting a neoplasm in a mammal with a virus comprising administering areplication-competent clonal RNA virus to said mammal wherein said virushas sensitivity to interferon.
 3. A method of treating a neoplasm in amammal comprising administering to said mammal a therapeuticallyeffective amount of an interferon-sensitive, replication-competentclonal RNA virus.
 4. A method as in claim 1 wherein said RNA virusreplicates at least 100-fold less in the presence of interferon comparedto in the absence of interferon.
 5. A method as in claim 1 wherein saidRNA virus replicates at least 1000-fold less in the presence ofinterferon compared to in the absence of interferon.
 6. A method as inclaim 1 wherein said administering step is systemic.
 7. A method as inclaim 1 wherein said neoplasm is a cancer.
 8. A method as in claim 1wherein said mammal is a human.
 9. A method as in claim 1 wherein saidclonal virus is plaque purified.
 10. A method as in claim 1 wherein saidclonal virus is of recombinant clonal origin.
 11. A method as in claim 1wherein said RNA virus is a Paramyxovirus.
 12. A method as in claim 11wherein said Paramyxovirus is purified to a level of at least 2×10⁹ PFUper mg of protein.
 13. A method as in claim 11 wherein saidParamyxovirus is purified to a level of at least 1×10¹⁰ PFU per mg ofprotein.
 14. A method as in claim 11 wherein said Paramyxovirus ispurified to a level of at least 6×10¹⁰ PFU per mg of protein.
 15. Amethod as in claim 11 wherein said Paramyxovirus is purified to a levelin which the particle per PFU ratio is no greater than
 5. 16. A methodas in claim 11 wherein said Paramyxovirus is purified to a level inwhich the particle per PFU ratio is no greater than
 3. 17. A method asin claim 11 wherein said Paramyxovirus is purified to a level in whichthe particle per PFU ratio is no greater than 1.2.
 18. A method as inclaim 11 wherein said Paramyxovirus is avian paramyxovirus type
 2. 19. Amethod as in claim 11 wherein said Paramyxovirus is NDV.
 20. A method asin claim 11 wherein said Paramyxovirus is mumps virus.
 21. A method asin claim 11 wherein said Paramyxovirus is human parainfluenza virus. 22.A method as claim 1 wherein said RNA virus is selected from the groupconsisting of a Rhabdoviris, Togavirus, Flavivirus, Reovirus,Picornavirus, and Coronavirus.
 23. A method as in claim 22 wherein saidTogavirus is Sindbis virus.
 24. A method as in claim 22 wherein saidReovirus has a modification at omega
 3. 25. A method as in claim 22wherein said Reovirus has an attenuating mutation at omega
 1. 26. Amethod as in claim 22 wherein said Reovirus is an attenuated rotavirus27. A method as in claim 26 wherein said rotavirus is rotavirus WC3. 28.A method of infecting a neoplasm in a mammal with a virus comprisingadministering an interferon-sensitive, replication-competent clonalvaccinia virus, having one or more mutations in one or more genesselected from the group consisting of K3L, E3L, and B18R, to saidmammal.
 29. A method of infecting a neoplasm in a mammal with a viruscomprising administering a replication-competent clonal vaccinia virus,having one or more mutations in one or more genes selected from thegroup consisting of K3L, E3L, and B18R, to said mammal wherein saidvirus has sensitivity to interferon.
 30. A method treating a neoplasm ina mammal comprising administering to said mammal a therapeuticallyeffective amount of an interferon-sensitive, replication-competentclonal vaccinia virus, having one or more mutations in one or more genesselected from the group consisting of K3L, E3L, and B18R.
 31. A methodas in claim 30 wherein said mammal is a human.
 32. A method as in claim30 wherein said vaccinia virus is a vaccinia virus having an attenuatingmutation in a gene selected from the group encoding vaccinia growthfactor, thymidine kinase, thymidylate kinase, DNA ligase, ribonucleotidereductase and dUTPase.
 33. A method of infecting a neoplasm in a mammalwith a virus comprising administering an interferon-sensitive,replication-competent clonal DNA virus, selected from the groupconsisting of Adenoviruses, Parvoviruses, Papovaviruses, andIridoviruses, to said mammal.
 34. A method of infecting a neoplasm in amammal with a virus comprising administering a replication-competentclonal DNA virus, selected from the group consisting of Adenoviruses,Parvoviruses, Papovaviruses, and Iridoviruses, to said mammal whereinsaid virus has sensitivity to interferon.
 35. A method of treating aneoplasm in a mammal comprising administering to said mammal atherapeutically effective amount of an interferon-sensitive, clonal DNAvirus selected from the group consisting of Adenoviruses, Parvoviruses,Papovaviruses, and Iridoviruses.
 36. A method as in claim 33 whereinsaid mammal is a human.
 37. A method as in claim 33 wherein saidAdenovirus virus has a modification in the VA1 transcripts causing saidAdenovirus to become interferon-sensitive.
 38. A method as in claim 37wherein said Adenovirus virus is selected from the group consisting ofvaccine strains of Ad-4, Ad-7 and Ad-21.
 39. A method of infecting aneoplasm in a mammal with a virus comprising administering aninterferon-sensitive, replication-competent clonal Herpesvirus to saidmammal.
 40. A method of infecting a neoplasm in a mammal with a viruscomprising administering a replication-competent clonal Herpesvirus tosaid mammal wherein said virus has sensitivity to interferon.
 41. Amethod treating a neoplasm in a mammal comprising administering to saidmammal a therapeutically effective amount of an interferon-sensitive,replication-competent clonal Herpesvirus.
 42. A method as in claim 41wherein said Herpesvirus is a member of the subfamily Betaherpesvirus orsubfamily Gammaherpesvirus.
 43. A method as in claim 41 wherein saidHerpesvirus is a member of the subfamily Alphaherpesvirus that is notHSV-1.
 44. A method as in claim 41 wherein said mammal is a human.
 45. Amethod as in claim 41 wherein said Herpesvirus is a member of thesubfamily Alphaherpesvirus which has decreased expression of the (2′-5′)An analog.
 46. A method as in claim 45 wherein said Herpesvirus is aHerpesvirus having an attenuating mutation selected from the groupconsisting of the genes encoding thymidine kinase, ribonucleotidereductase, or a deletion in the b′a′c′ inverted repeat locus.
 47. Amethod as in claim 45 wherein said Herpesvirus has a modification in thegamma 34.5 gene.
 48. A method as in claim 41 wherein said Herpesvirushas a modification in the gamma 34.5 gene and an attenuating mutation inthe gene encoding of thymidine kinase, or a deletion in the b′a′c′inverted repeat locus or functionally analogous loci.
 49. A method as inclaim 41 wherein said Herpesvirus is a Herpesvirus having an attenuatingmutation in a gene selected from the group consisting of thymidinekinase, and ribonucleotide reductase, or a deletion in the b′a′c′inverted repeat locus.
 50. A method as in claim 1 wherein said neoplasmis a cancer selected from the group consisting of lung, colon, prostate,breast and brain cancer.
 51. A method as in claim 1 wherein saidneoplasm is a solid tumor.
 52. A method as in claim 50 wherein saidbrain cancer is a glioblastoma.
 53. A method as in claim 1 wherein saidvirus contains a gene encoding interferon to permit the viral expressionof interferon.
 54. A method as in claim 1 wherein said virus contains agene encoding a pro-drug activating enzyme.
 55. A method as in claim 1further comprising administering IFN, before, during or afteradministration of said virus.
 56. A method as in claim 55 wherein saidinterferon is selected from the group consisting of α-IFN, β-IFN, ω-IFN,γ-IFN, and synthetic consensus forms of IFN.
 57. A method as in claim 1further comprising administering a tyrosine kinase inhibitor before,during or after administration of said virus.
 58. A method as in claim 1further comprising administering a compound selected from the group ofcompounds comprising a purine nucleoside analog, tyrosine kinaseinhibitor, cimetidine, and mitochondrial inhibitor.
 59. A method as inclaim 1 further comprising administering a chemotherapeutic agentbefore, during or after administration of said virus.
 60. A method as inclaim 1 further comprising administering a cytokine before, during orafter administration of said virus.
 61. A method as in claim 1 furthercomprising administering an immunosuppresant before, during or afteradministration of said virus.
 62. A method as in claim 1 furthercomprising administering a viral replication controlling amount of acompound selected from the group consisting of IFN, ribavirin,acyclovir, and ganciclovir.
 63. A method as in claim 1 wherein saidadministration is intravenous or intratumoral.
 64. A method of infectinga neoplasm which is at least 1 centimeter in size in a mammal with avirus comprising administering a clonal virus, selected from the groupconsisting of (1) RNA viruses; (2) Hepadenavirus; (3) Parvovirus; (4)Papovavirus; (5) Herpesvirus; (6) Poxvirus; and (7) Iridovirus, to saidmammal.
 65. A method of treating a neoplasm in a mammal, comprisingadministering to said mammal a therapeutically effective amount of aclonal virus selected from the group consisting of (1) RNA virus; (2)Hepadenavirus; (3) Parvovirus; (4) Papovavirus; (5) Herpesvirus; (6)Poxvirus; and (7) Iridovirus, wherein said neoplasm is at least 1centimeter in size.
 66. A method as in claim 64, wherein said neoplasmis at least 300 mm³ in volume.
 67. A method as in claim 64, wherein saidRNA virus is a Paramyxovirus.
 68. A method as in claim 67, wherein saidParamyxovirus is NDV.
 69. A method as in claim 64, wherein said mammalis a human.
 70. A method as in claim 64, wherein said administration isintravenous or intratumoral.
 71. A method as in claim 64, wherein saidparamyxovirus is purified to a level of at least 2×10⁹ PFU per mgprotein.
 72. A method as in claim 68, wherein said NDV is mesogenic. 73.A method as in claim 65 wherein said neoplasm is cancerous.
 74. A methodof treating a tumor in a mammal, comprising administering to said mammala therapeutically effective amount of an RNA virus cytocidal to saidtumor, wherein said mammal has a tumor burden comprising at least 1.5%of the total body weight of said mammal.
 75. A method as in claim 74,wherein said tumor does not respond to chemotherapy.
 76. A method ofscreening tumor cells or tissue freshly removed from the patient todetermine the sensitivity of said cells or tissue to killing by a viruscomprising subjecting a tissue sample to a differential cytotoxicityassay using an interferon-sensitive virus.
 77. A method as in claim 76further comprising the step of screening said cells or tissue forprotein, or mRNA encoding protein, selected from the group consisting ofp68 protein kinase, C-Myc, C-Myb, ISGF-3, IRF-1, IFN receptor, and p58.78. A method for identifying a virus with antineoplastic activity in amammal comprising: a) using said test virus to infect i) cells deficientin an interferon-mediated antiviral activity, and ii) cells competent inan interferon-mediated antiviral activity, and b) determining whethersaid test virus kills said cells deficient in an interferon-mediatedantiviral activity preferentially to said cells competent ininterferon-mediated antiviral activity.
 79. A method as in claim 78wherein said cells deficient in an interferon-mediated antiviralactivity are KB human head and neck carcinoma cells.
 80. A method as inclaim 78 wherein said cells competent in an interferon-mediatedantiviral activity are human skin fibroblasts.
 81. A method of makingviruses for use in antineoplastic therapy comprising: (a) modifying anexisting virus by diminishing or ablating a viral mechanism for theinactivation of the antiviral effects of IFN, and optionally (b)creating an attenuating mutation
 82. A method of controlling viralreplication in a mammal treated with a virus selected from the groupconsisting of RNA viruses, Adenoviruses, Poxviruses, Iridoviruses,Parvoviruses, Hepadnaviruses, Varicellaviruses, Betaherpesviruses, andGammaherpesviruses comprising administering an antiviral compound.
 83. Amethod as in claim 82 wherein said antiviral compound is interferon. 84.A method as in claim 82 wherein said antiviral is selected from thegroup consisting of ribavirin, acyclovir, and ganciclovir.
 85. A methodas in claim 82 wherein said antiviral is a neutralizing antibody to saidvirus.
 86. A Paramyxovirus purified by ultracentrifugation withoutpelleting.
 87. A Paramyxovirus purified to a level of at least 2×10⁹PFU/mg protein.
 88. A Paramyxovirus as in claim 87 wherein saidparamyxovirus is grown in eggs and is substantially free ofcontaminating egg proteins.
 89. A Paramyxovirus as in claim 87 whereinsaid paramyxovirus has a particle per PFU ratio no greater than
 5. 90. AParamyxovirus as in claim 87 wherein said paramyxovirus has a particleper PFU ratio no greater than
 3. 91. A Paramyxovirus as in claim 87wherein said paramyxovirus has a particle per PFU ratio no greater than1.2.
 92. A Paramyxovirus purified to a level of at least 1×10¹⁰ PFU/mgprotein.
 93. A Paramyxovirus purified to a level of at least 6×10¹⁰PFU/mg protein.
 94. A Paramyxovirus as in claim 87 wherein said virus iscytocidal.
 95. A Paramyxovirus as in claim 87 wherein said Paramyxovirusis Newcastle disease virus.
 96. An NDV as in claim 95 wherein said NDVis cytocidal.
 97. An NDV as in claim 95 wherein said NDV is mesogenic.98. An RNA virus purified to a level of at least 2×10⁹ PFU/mg protein.99. An RNA virus as in claim 98 wherein said virus isreplication-competent.
 100. A replication-competent cytocidal viruswhich is interferon-sensitive and purified to a level of at least 2×10⁹PFU/mg protein.
 101. A cytocidal virus as in claim 100 wherein saidvirus is clonal.
 102. A cytocidal DNA virus which isinterferon-sensitive and purified to a level of at least 2×10⁹ PFU/mgprotein.
 103. A cytocidal DNA virus as in claim 102 wherein said virusis a Poxvirus.
 104. A Poxvirus as in claim 103 wherein said Poxvirus isa vaccinia virus having one or more mutations in one or more genesselected from the group consisting of K3L, E3L, and B18R.
 105. Areplication-competent vaccinia virus having a) one or more mutations inone or more of the K3L, E3L and B18R genes, and b) an attentuatingmutation in one or more of the genes encoding thymidine kinase,ribonucleotide reductase, vaccinia growth factor, thymidylate kinase,DNA ligase, dUTPase.
 106. A replication-competent vaccinia virus havingone or more mutations in two or more genes selected from the groupconsisting of K3L, E3L, and B18R.
 107. A Herpesvirus having amodification in the expression of the (2′-5′) A analog.
 108. A Reovirushaving a mutation at omega 3 and purified to a level of at least 2×10⁹PFU/mg protein
 109. A Reovirus having mutations at omega 1 and omega 3.110. A method of purifying an RNA virus comprising the steps of: a)generating a clonal virus, and b) purifying said clonal virus byultracentrifugation without pelleting.
 111. A method as in claim 110wherein said RNA virus is replication-competent.
 112. A method ofpurifying a Paramyxovirus comprising purifying said virus byultracentrifugation without pelleting.
 113. A method as in claim 112wherein said purifying step additionally comprises prior to saidultracentrifugation: a) plaque purifying to generate a clonal virus, b)inoculating eggs with said clonal virus, c) incubating said eggs, d)chilling said eggs, e) harvesting allantoic fluid from said eggs and, f)removing cell debris from said allantoic fluid.
 114. A method as inclaim 112 wherein said Paramyxovirus virus is NDV.
 115. A method ofinfecting a neoplasm in a mammal with a virus comprising administeringan interferon-sensitive, replication-competent RNA virus to said mammal.116. A method as in claim 1 wherein said virus is selected from thegroup consisting of the Newcastle disease virus strain MK107, Newcastledisease virus strain NJRoakin, Sindbis virus, and Vesicular stomatitisvirus.
 117. A method of infecting a neoplasm in a mammal with a viruscomprising administering a clonal virus selected from the groupconsisting of the Newcastle disease virus strain MK107, Newcastledisease virus strain NJRoakin, Sindbis virus, and Vesicular stomatitisvirus.
 118. A method as in claim 1 or claim 28 or claim 33 wherein saidvirus is administered as more than one dose.
 119. A method as in claim118 wherein the first dose is a desensitizing dose.
 120. A method as inclaim 119 wherein said first dose is administered intravenously and asubsequent dose administered intravenously.
 121. A method as in claim119 wherein said first dose is administered intravenously and asubsequent dose administered intraperitoneally.
 122. A method as inclaim 119 wherein said first dose is administered intravenously and asubsequent dose administered intra-arterially.
 123. A method of treatinga neoplasm in a mammal comprising subjecting a sample from said mammalto an immunoassay to detect the amount of virus receptor present, and ifsaid receptor is present, administering an interferon-sensitive,replication competent clonal virus, which bind said receptor, to saidmammal.
 124. A method as in claim 123 wherein said virus is Sindbis andsaid receptor is the high affinity laminin receptor.
 125. A method as inclaim 1 or claim 28 or claim 33 wherein said virus is administered overthe course of at least 4 minutes.
 126. A method of treating tumorascites comprising administering an interferon-sensitive, replicationcompetent clonal virus.
 127. A method of reducing pain in a mammalcomprising administering an interferon-sensitive, replication competentclonal virus.